The present invention relates to a compound, a liposome comprising said compound, a virus-like particle comprising said compound, a pharmaceutical composition comprising or consisting of said compound, said liposome or said virus-like particle, a kit comprising or consisting of said compound, said liposome, said virus-like particle or said pharmaceutical composition and use of the compound, liposome, virus-like particle or pharmaceutical composition in the treatment of a disease characterized by overexpression of fibroblast activation protein (FAP).

Background of the Invention

Tumor growth and spread are not only determined by the cancer cells, but also by the non-malignant constituents of the malignant lesion, which are subsumed under the term stroma. The stroma may represent over 90% of the tumor mass in tumors with desmoplastic reaction such as breast, colon and pancreatic carcinoma. Especially a subpopulation of fibroblasts called cancer-associated fibroblasts (CAFs) is known to be involved in tumor growth, migration and progression. Therefore, these cells represent an attractive target for anti-tumor therapy.

A distinguishing feature of CAFs is the expression of seprase or fibroblast activation protein a (FAP-a), a type II membrane bound glycoprotein belonging to the dipeptidyl peptidase 4 (DPP4) family. FAP-a has both dipeptidyl peptidase and endopeptidase activity. The endopeptidase activity distinguishes FAP-a from the other members of the DPP4 family. Identified substrates for the endopeptidase activity so far are denatured Type I collagen, al- antitrypsin and several neuropeptides. FAP-a has a role in normal developmental processes during embryogenesis and in tissue modelling. It is not or only at insignificant levels expressed on adult normal tissues. However, high expression occurs in wound healing, arthritis, artherosclerotic plaques, fibrosis and in more than 90% of epithelial carcinomas.

The appearance of FAP-a in CAFs in many epithelial tumors and the fact that overexpression is associated with a worse prognosis in cancer patients led to the hypothesis that FAP-a activity is involved in cancer development as well as in cancer cell migration and spread. Therefore, the targeting of this enzyme for therapy can be considered as a promising strategy for the treatment of malignant tumors. The present inventors developed a small molecule therapeutic based on a FAP-a specific inhibitor. Furthermore, the molecules can be used for the treatment of non-malignant diseases such as chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and keloid disorders. Summary of the Invention

In a first aspect, the present invention provides a compound of Formula (I)

wherein

Q, R, U, V, W, Y, Z are individually present or absent under the proviso that at least three of Q, R, U, V, W, Y, Z are present;

Q, R, U, V, W, Y, Z are independently selected form the group consisting of O, CH ₂ , NR ⁴ , C=0, C=S, C=NR ⁴ , HCR ⁴ and R ⁴ CR ⁴ , with the proviso that two Os are not directly adjacent to each other;

R ¹ and R ² are independently selected from the group consisting of -H, -OH, halo, Ci-6-alkyl, -

O-Ci-6-alkyl, S-Ci-e-alkyl;

R ³ is selected from the group consisting of -H , -CN , -B(OH) ₂ , -C(O) -alkyl, -C(O) -aryl-, - C=C-C(0) -aryl, -C=C-S(0) ₂ -aryl, -C0 ₂ H , -SOsH , -S0 ₂ NH ₂ ,-P0 ₃ H ₂ , and 5-tetrazolyl;

R ⁴ is selected from the group consisting of -H, -Ci-6-alkyl, -O-Ci-6-alkyl, -S-Ci-6-alkyl, aryl, and -Ci-6-aralkyl, each of said -Ci-6-alkyl being optionally substituted with from 1 to 3 substituents selected from -OH, oxo, halo and optionally connected to Q, R, U, V, W, Y or Z; R ⁵ is selected from the group consisting of -H, halo and Ci-6-alkyl;

R ⁶ , and R ⁷ are independently selected from the group consisting of-H, an , under the proviso that R ⁶ and R ⁷ are not at the same time H,

wherein L is a linker,

wherein D, A, E, and B are individually present or absent, preferably wherein at least A, E, and B are present, wherein when present:

D is a linker;

A is selected from the group consisting of NR ⁴ , O, S, and CH ₂ ;

E is selected from the group consisting of

wherein i is 1, 2, or 3;

wherein j is 1, 2, or 3;

wherein k is 1, 2, or 3;

wherein m is 1, 2, or 3;

B is selected from the group consisting of S, NR ⁴ , NR ⁴ -0, NR ⁴ -Ci-6-alkyl, NR ⁴ -Ci-6-alkyl-NR ⁴ , and a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein NR ⁴ -Ci-6-alkyl-NR ⁴ and the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of Ci-6-alkyl, aryl, Ci-6-aralkyl; and;

R ⁸ is selected from the group consisting a cytostatic and/or cytotoxic agent, a cytokine, an immunomodulatory molecule, an amphiphilic substance, polyglycolic acid, polylactic acid or a derivative thereof, a nucleic acid, a viral structural protein, a protein, biotin and combinations thereof; is a l-naphtyl moiety or a 5 to 10- membered N-containing aromatic or non aromatic mono- or bicyclic heterocycle, wherein there are 2 ring atoms between the N atom and X; said heterocycle optionally further comprising 1, 2 or 3 heteroatoms selected from O, N and S; and X is a C atom;

or a pharmaceutically acceptable tautomer, racemate, hydrate, solvate, or salt thereof

In a second aspect, the present invention relates to a liposome comprising the compound of the first aspect.

In a third aspect, the present invention relates to a virus-like particle (VLP) comprising the compound of the first aspect.

In a fourth aspect, the present invention relates to a pharmaceutical composition comprising or consisting of at least one compound of the first aspect, or the liposome of the second aspect, or the virus-like particle (VLP) of the third aspect, and, optionally, a pharmaceutically acceptable carrier and/or excipient.

In a fifth aspect, the present invention relates to the compound of the first aspect, or the liposome of the second aspect, or the virus-like particle (VLP) of the third aspect, or the pharmaceutical composition of the fourth aspect for use in the treatment of a disease characterized by overexpression of fibroblast activation protein (FAP) in an animal or a human subject.

In a sixth aspect, the present invention relates to a kit comprising or consisting of the compound of the first aspect, or the liposome of the second aspect, or the virus-like particle (VLP) of the third aspect, or the pharmaceutical composition of the second aspect and instructions for the treatment of a disease.

List of Figures

In the following, the content of the figures comprised in this specification is described. In this context please also refer to the detailed description of the invention above and/or below.

Figure 1: In vitro characterization of ¹²⁵ I-FAPI-01 and ¹⁷⁷ Lu-FAPI-02.

A. Binding of radiolabeled FAPI-01 and FAPI-02 to different human cancer cell lines as well as cell lines transfected with human FAP-a (HT-1080-FAP), murine FAP-a (HEK-muFAP) and human CD26 (HEK-CD26) after 60 min of incubation. B. Internalization of radiolabeled F API-01 and FAPI-02 into HT-1080-FAP cells after incubation for 10 min to 24 h. The internalized proportion is shown in grey and black, respectively; the extracellularly bound fraction is indicated by the white bars. C. Competitive binding of radiolabeled FAPI-01 and FAPI-02 to HT-1080-FAP cells after adding increasing concentrations of unlabeled FAPI-01 and Fu-F API-02. D. Internalization of FAPI-02 into FAP-a positive and negative cell lines. Blue: DAPI; green: FAPI-02-Atto488. E+F. Efflux kinetics of FAPI-01 and FAPI-02 after 1 h incubation of HT-1080-FAP cells with radiolabeled compounds followed by incubation with compound-free medium for 1 to 24 h. All values are given as percentage of total applied dose normalized to 1 million cells (%ID/l mio cells).

Figure 2: Binding specificity and relative internalization rates of FAPI derivatives. A-C. Binding and internalization rates of FAPI-03 to FAPI- 15 in relation to FAPI-02 (defined as 100%). Internalization rates after 1, 4 and 24 hrs of incubation are depicted in grey; the extracellular bound fraction is represented by the white bars. D. Binding of selected FAPI derivatives to HEK cells expressing murine FAP-a and human CD26 after 60 min of incubation. Right side : Ratio of muFAP to CD26 binding. E. Competitive binding of selected FAPI derivatives to HT-1080-FAP cells after adding increasing concentrations of unlabeled compound.

Detailed Descriptions of the Invention

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Throughout this specification and the claims, which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being optional, preferred or advantageous may be combined with any other feature or features indicated as being optional, preferred or advantageous.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as being“incorporated by reference” . In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments; however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Definitions

In the following, some definitions of terms frequently used in this specification are provided. These terms will, in each instance of its use, in the remainder of the specification have the respectively defined meaning and preferred meanings.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise.

In the following definitions of the terms: alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, alkenyl and alkynyl are provided. These terms will in each instance of its use in the remainder of the specification have the respectively defined meaning and preferred meanings.

The term“alkyl” refers to a saturated straight or branched carbon chain. Preferably, the chain comprises from 1 to 10 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 e.g. methyl, ethyl methyl, ethyl, propyl, /so- propyl, butyl, /v -butyl, /er/-butyl, pentyl, hexyl, pentyl, or octyl. Alkyl groups are optionally substituted.

The term “heteroalkyl” refers to a saturated straight or branched carbon chain. Preferably, the chain comprises from 1 to 9 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 e.g. methyl, ethyl, propyl, iso- propyl, butyl, v -butyl, sec-butyl, tert- butyl, pentyl, hexyl, pentyl, octyl, which is interrupted one or more times, e.g. 1 , 2, 3, 4, 5, with the same or different heteroatoms. Preferably the heteroatoms are selected from O, S, and N, e.g. -O-CH3, -S-CH3, -CH2-O-CH3, -CH2-O-C2H5, -CH2-S-CH3, -CH2-S-C2H5, -C2H4-O-CH3, -C2H4-O-C2H5, -C2H4-S-CH3, - C2H4-S-C2H5 etc. Heteroalkyl groups are optionally substituted.

The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl", respectively, with preferably 3, 4, 5, 6, 7, 8, 9 or 10 atoms forming a ring, e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl etc. The terms "cycloalkyl" and "heterocycloalkyl" are also meant to include bicyclic, tricyclic and polycyclic versions thereof. The term“heterocycloalkyl” preferably refers to a saturated ring having five of which at least one member is a N, O or S atom and which optionally contains one additional O or one additional N; a saturated ring having six members of which at least one member is a N, O or S atom and which optionally contains one additional O or one additional N or two additional N atoms; or a saturated bicyclic ring having nine or ten members of which at least one member is a N, O or S atom and which optionally contains one, two or three additional N atoms. “Cycloalkyl” and “heterocycloalkyl” groups are optionally substituted. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, l-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, spiro[3,3]heptyl, spiro[3,4]octyl, spiro[4,3]octyl, spiro[3,5]nonyl, spiro[5,3]nonyl, spiro[3,6]decyl, spiro[6,3]decyl, spiro[4,5]decyl, spiro[5,4]decyl, bicyclo[2.2.l]heptyl, bicyclo[2.2.2]octyl, adamantyl, and the like. Examples of heterocycloalkyl include l-(l,2,5,6-tetrahydropyridyl), 1- piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, 1,8 diazo-spiro-[4,5] decyl, 1,7 diazo-spiro-[4,5] decyl, 1,6 diazo-spiro-[4,5] decyl, 2,8 diazo-spiro[4,5] decyl, 2,7 diazo-spiro[4,5] decyl, 2,6 diazo-spiro[4,5] decyl, 1,8 diazo-spiro-[5,4] decyl, 1,7 diazo-spiro- [5,4] decyl, 2,8 diazo-spiro-[5,4] decyl, 2,7 diazo-spiro[5,4] decyl, 3,8 diazo-spiro[5,4] decyl, 3,7 diazo-spiro[5,4] decyl, l-azo-7,l l-dioxo-spiro[5,5] undecyl, l,4-diazabicyclo[2.2.2]oct-2- yl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1- piperazinyl, 2-piperazinyl, and the like.

The term“aryl” preferably refers to an aromatic monocyclic ring containing 6 carbon atoms, an aromatic bicyclic ring system containing 10 carbon atoms or an aromatic tricyclic ring system containing 14 carbon atoms. Examples are phenyl, naphtyl or anthracenyl. The aryl group is optionally substituted.

The term“aralkyl” refers to an alkyl moiety, which is substituted by aryl, wherein alkyl and aryl have the meaning as outlined above. An example is the benzyl radical. Preferably, in this context the alkyl chain comprises from 1 to 8 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, or 8, e.g. methyl, ethyl methyl, ethyl, propyl, /A -propyl, butyl, Ao-butyl, sec-butenyl, / _< °r/-butyl, pentyl, hexyl, pentyl, octyl. The aralkyl group is optionally substituted at the alkyl and/or aryl part of the group.

The term“heteroaryl” preferably refers to a five or six-membered aromatic monocyclic ring wherein at least one of the carbon atoms are replaced by 1 , 2, 3, or 4 (for the five membered ring) or 1, 2, 3, 4, or 5 (for the six membered ring) of the same or different heteroatoms, preferably selected from O, N and S; an aromatic bicyclic ring system wherein 1, 2, 3, 4, 5, or 6 carbon atoms of the 8, 9, 10, 11 or 12 carbon atoms have been replaced with the same or different heteroatoms, preferably selected from O, N and S; or an aromatic tricyclic ring system wherein 1, 2, 3, 4, 5, or 6 carbon atoms of the 13 , 14, 15 , or 16 carbon atoms have been replaced with the same or different heteroatoms, preferably selected from O, N and S. Examples are oxazolyl, isoxazolyl, l,2,5-oxadiazolyl, l,2,3-oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, l,2,3-triazolyl, thiazolyl, isothiazolyl, l,2,3,-thiadiazolyl, l,2,5-thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, l,2,3-triazinyl, l,2,4-triazinyl, l,3,5-triazinyl, l-benzofuranyl, 2- benzofuranyl, indoyl, isoindoyl, benzothiophenyl, 2-benzothiophenyl, lH-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, 2,l-benzosoxazoyl, benzothiazolyl, 1,2- benzisothiazolyl, 2,l-benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, quinolinyl, l,2,3-benzotriazinyl, or l,2,4-benzotriazinyl.

The term“heteroaralkyl” refers to an alkyl moiety, which is substituted by heteroaryl, wherein alkyl and heteroaryl have the meaning as outlined above. An example is the 2- alklypyridinyl, 3-alkylpyridinyl, or 2-methylpyridinyl. Preferably, in this context the alkyl chain comprises from 1 to 8 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, or 8, e.g. methyl, ethyl methyl, ethyl, propyl, / ^' .so- propyl, butyl, Ao-butyl, sec-butenyl, tert- butyl, pentyl, hexyl, pentyl, octyl. The heteroaralkyl group is optionally substituted at the alkyl and/or heteroaryl part of the group.

The terms“alkenyl” and“cycloalkenyl” refer to olefinic unsaturated carbon atoms containing chains or rings with one or more double bonds. Examples are propenyl and cyclohexenyl. Preferably, the alkenyl chain comprises from 2 to 8 carbon atoms, i.e. 2, 3, 4, 5, 6, 7, or 8, e.g. ethenyl, l-propenyl, 2-propenyl, A -propcnyl, l-butenyl, 2-butenyl, 3-butenyl, Ao-butenyl, sec-butenyl, l-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, hexenyl, pentenyl, octenyl. Preferably the cycloalkenyl ring comprises from 3 to 8 carbon atoms, i.e. 3, 4, 5, 6, 7, or 8, e.g. l-cyclopropenyl, 2-cyclopropenyl, l-cyclobutenyl, 2-cylcobutenyl, l-cyclopentenyl, 2-cyclopentenyl, 3-cyclopentenyl, cyclohexenyl, cyclopentenyl, cyclooctenyl.

The term“alkynyl” refers to unsaturated carbon atoms containing chains or rings with one or more triple bonds. An example is the propargyl radical. Preferably, the alkynyl chain comprises from 2 to 8 carbon atoms, i.e. 2, 3, 4, 5, 6, 7, or 8, e.g. ethynyl, l-propynyl, 2- propynyl, l-butynyl, 2-butynyl, 3-butynyl, l-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, hexynyl, pentynyl, octynyl.

In one embodiment, carbon atoms or hydrogen atoms in alkyl, heteroalkyl, cycloalkyl, aryl, aralkyl, alkenyl, cycloalkenyl, alkynyl radicals may be substituted independently from each other with one or more elements selected from the group consisting of O, S, N or with groups containing one or more elements selected from the group consisting of O, S, N. Embodiments include alkoxy, cycloalkoxy, arykoxy, aralkoxy, alkenyloxy, cycloalkenyloxy, alkynyloxy, alkylthio, cycloalkylthio, arylthio, aralkylthio, alkenylthio, cycloalkenylthio, alkynylthio, alkylamino, cycloalkylamino, arylamino, aralkylamino, alkenylamino, cycloalkenylamino, alkynylamino radicals.

Other embodiments include hydroxyalkyl, hydroxycycloalkyl, hydroxyaryl, hydroxyaralkyl, hydroxyalkenyl, hydroxycycloalkenyl, hydroxyalinyl, mercaptoalkyl, mercaptocycloalkyk, mercaptoaryl, mercaptoaralkyl, mercaptoalkenyl, mercaptocycloalkenyl, mercaptoalkynyl, aminoalkyl, aminocycloalkyl, aminoaryl, aminoaralkyl, aminoalkenyl, aminocycloalkenyl, aminoalkynyl radicals.

In another embodiment, hydrogen atoms in alkyl, heteroalkyl, cycloalkyl, aryl, aralkyl, alkenyl, cycloalkenyl, alkynyl radicals may be substituted independently from each other with one or more halogen atoms. One radical is the trifluoromethyl radical.

If two or more radicals or two or more residues can be selected independently from each other, then the term“independently” means that the radicals or the residues may be the same or may be different.

As used herein a wording defining the limits of a range of length such as, e. g.,“from 1 to 6” means any integer from 1 to 6, i. e. 1, 2, 3, 4, 5 and 6. In other words, any range defined by two integers explicitly mentioned is meant to comprise and disclose any integer defining said limits and any integer comprised in said range.

The term“halo” as used herein refers to a halogen residue selected from the group consisting of F, Br, I and Cl. Preferably, the halogen is F.

The term“linker” as used herein refers to any chemically suitable linker. Preferably, linker are not or only slowly cleaved under physiological conditions. Thus, it is preferred that the linker does not comprise recognition sequences for proteases or recognition structures for other degrading enzymes. Since it is preferred that the compounds of the invention are administered systemically to allow broad access to all compartments of the body and subsequently enrichment of the compounds of the invention wherever in the body the tumor is located, it is preferred that the linker is chosen in such that it is not or only slowly cleaved in blood. The cleavage is considered slowly, if less than 50% of the linkers are cleaved 2 h after administration of the compound to a human patient. Suitable linkers, for example, comprises or consists of optionally substituted alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, aralkyl, heteroaralyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, sulfonyl, amines, ethers, thioethers phosphines, phosphoramidates, carboxamides, esters, imidoesters, amidines, thioesters, sulfonamides, 3-thiopyrrolidine-2,5-dion, carbamates, ureas, guanidines, thioureas, disulfides, oximes, hydrazines, hydrazides, hydrazones, diaza bonds, triazoles, triazolines, tetrazines, platinum complexes and amino acids, or combinations thereof. Preferably, the linker comprises or consists of 1 ,4-piperazine, 1, 3-propane and a phenolic ether or combinations thereof

The linker can also be a cleavable linker such as a peptide motif that is cleaved by cathepsin. Any suitable linker that is cleavable by cathepsin can be used. Suitable cleavable peptide linkers are disclosed in Peterson et af, Bioconjugate Chem., 1998. Suitable cleavable linkers, for example, comprises or consists of optionally substituted NC Tyr-Gln-Gly-Val-Gln-Phe- Lys(Aminobenzoyl), N02Tyr-Asn-Gly-Thr-Gly-Phe-Lys(Aminobenzoyl), NChTyr-Ser-Val- Val-Phe-Phe-Lys(Aminobenzoyl), NChTyr-Val-Gln-Ser-Ala-Phe, Multiple-Val-Gln-Phe-Val, NCteTyr-Gly-Val-Phe-Gln-Phe, N02Tyr-Gly-Thr-Val-Ala-Phe-Lys(Aminobenzoyl), NCteTyr- Ala-Thr-Ala-Phe-Phe-Lys(Aminobenzoyl), NChTyr-Gly-Ser-Val-Gln-Phe-

Lys(Aminobenzoyl), N02Tyr-Gly-Gly-Gln-Phe-Phe-Lys(Aminobenzoyl), NChTyr-Gln-Ser- Val-Gly-Phe-Lys(Aminobenzoyl), N02Tyr-Gly-Ser-Thr-Phe-Phe-Lys(Aminobenzoyl),

N02Tyr-Gly-Thr-Val-Gln-Phe-Lys(Aminobenzoyl), NChTyr-Gly-Ser-Thr-Phe-Phe-

Lys(Aminobenzoyl), N02Tyr-Gly-Val-Ala-Gly-Phe-Lys(Aminobenzoyl), NChTyr-Gly-Ser- Thr-Phe-Phe-Lys(Aminobenzoyl), N02Tyr-Ala-Ala-Gly-Thr-Phe-Lys(Aminobenzoyl),

N02Tyr-Val-Ala-Gln-Phe, N02Tyr-Gln-Gly-Val-Gly-Phe-Lys(Aminobenzoyl), NCteTyr-Val- Asn-Asn-Asn-Phe-Lys(Aminobenzoyl), N02Tyr-Ala-Ser-Ala-Asn-Phe-Lys(Aminobenzoyl), N02Tyr-Phe-Gln-Thr-Gln-Phe-Lys(Aminobenzoyl), NChTyr-Ala-Ala-Ala-Ser-Phe-

Lys(Aminobenzoyl), N02Tyr-Gln-Tyr-Ser-Gly-Phe-Lys(Aminobenzoyl), NChTyr-Ala-Ala- Thr-Ala-Phe-Lys(Aminobenzoyl), N02Tyr-Ala-Thr-Gln-Phe-Phe-Lys(Aminobenzoyl),

N02Tyr-Gln-Ser-Ala-Ser-Phe-Lys(Aminobenzoyl), NChTyr-Gly-Thr-Ser-Phe-Phe-

Lys(Aminobenzoyl), N02Tyr-Thr-Ala-Gly-Ala-Phe-Lys(Aminobenzoyl), NChTyr-Ala-Thr- Thr-Phe-Phe-Lys(Aminobenzoyl), N02Tyr-Ala-Ser-Gly-Ser-Phe-Lys(Aminobenzoyl),

N02Tyr-Gly-Thr-Thr-Phe-Phe-Lys(Aminobenzoyl), NChTyr-Gly-Ala-Ala-Gly-Phe-

Lys(Aminobenzoyl), N02Tyr-Gly-Thr-Gln-Phe-Phe-Lys(Aminobenzoyl), NChTyr-Ala-Ala- Thr-Gly-Phe-Lys(Aminobenzoyl), N02Tyr-Gly-Thr-Gln-Phe-Phe-Lys(Aminobenzoyl), N02Tyr-Gln-Thr-Val-Gly-Phe-Lys(Aminobenzoyl), NChTyr-Gly-Thr-Gln-Phe-Phe-

Lys(Aminobenzoyl), N02Tyr-Ala-Ser-Ala-Gly-Phe-Lys(Aminobenzoyl), NChTyr-Gly-Gln- Ser-Phe-Phe-Lys(Aminobenzoyl), N02Tyr-Thr-Ser-Ala-Thr-Phe-Lys(Aminobenzoyl),

N02Tyr-Gly-Thr-Val-Ala-Phe-Lys(Aminobenzoyl), NChTyr-Thr-Ala-Gln-Ala-Phe-

Lys(Aminobenzoyl), N02Tyr-Gly-Val-Ala-Ala-Phe-Lys(Aminobenzoyl), NChTyr-Val-Ala- Ser-Ala-Phe-Lys(Aminobenzoyl), N02Tyr-Gln-Gly-Ser-Phe-Phe-Lys(Aminobenzoyl), N02Tyr-Thr-Ala-Thr-Asn-Phe-Lys(Aminobenzoyl), NCkTyr-Ala-Thr-Ser-Phe-Phe-

Lys(Aminobenzoyl), N02Tyr-Thr-Gly-Val-Gly-Phe-Lys(Aminobenzoyl), NCteTyr-Gly-Thr- Ala-Phe-Phe-Lys(Aminobenzoyl), N02Tyr-Gln-Val-Ala-Gly-Phe-Lys(Aminobenzoyl), N02Tyr-Gly-Ser-Ala-Gln-Phe-Lys(Aminobenzoyl), NCkTyr-Val-Ala-Ala-Gln-Phe-

Lys(Aminobenzoyl), N02Tyr-Gln-Thr-Ala-Thr-Phe-Lys(Aminobenzoyl), NCteTyr-Thr-Gly- Tyr-Thr-Phe-Lys(Aminobenzoyl), N02Tyr-Ser-Ala-Gly-Thr-Phe-Lys(Aminobenzoyl),

N02Tyr-Val-Tyr-Tyr-Val-Phe, NCkTyr-Ala-Ser-Tyr-Gly-Phe, Z-Phe-Lys-PABC, Z-Phe-Lys, Z-Val-Lys-PABC, Z-Ala-Lys-P ABC , Phe-Phe-Lys-PABC, D-Phe-Phe-Lys-PABC, D-Ala- Phe-Lys-PABC, Gly-Phe-Lys-PABC, Ac-Phe-Lys-PABC, HCO-Phe-Lys-PABC, Phe-Lys- PABC, Z-Lys-PABC, Z-Val-Cit-PABC, Z-Val-Cit, Z-Phe-Cit-PABC, Z-Leu-Cit-PABC, Z-Ile- Cit-PABC, Z-Trp-Cit-PABC, Z-Phe-Arg(N02)-PABC and Z-Phe-Arg(Ts)-PABC.

The expression“optionally substituted” refers to a group in which one, two, three or more hydrogen atoms may have been replaced independently of each other by the respective substituents.

As used herein, the term "amino acid" refers to any organic acid containing one or more amino substituents, e.g. a-, b- or g-amino, derivatives of aliphatic carboxylic acids. In the polypeptide notation used herein, e.g. Xaa5, i.e. XaalXaa2Xaa3Xaa4Xaa5, wherein Xaal to Xaa5 are each and independently selected from amino acids as defined, the left hand direction is the amino terminal direction and the right hand direction is the carboxy terminal direction, in accordance with standard usage and convention.

The term "conventional amino acid" refers to the twenty naturally occurring amino acids, and encompasses all stereomeric isoforms, i.e. D,L-, D- and L-amino acids thereof. These conventional amino acids can herein also be referred to by their conventional three- letter or one-letter abbreviations and their abbreviations follow conventional usage (see, for example, Immunology— A Synthesis, 2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland Mass. (1991)).

The term "non-conventional amino acid" refers to unnatural amino acids or chemical amino acid analogues, e.g. a,a-disubstituted amino acids, N-alkyl amino acids, homo-amino acids, dehydroamino acids, aromatic amino acids (other than phenylalanine, tyrosine and tryptophan), and ortho-, meta- or para-aminobenzoic acid. Non-conventional amino acids also include compounds which have an amine and carboxyl functional group separated in a 1,3 or larger substitution pattern, such as b-alanine, g-amino butyric acid, Freidinger lactam, the bicyclic dipeptide (BTD) , amino-methyl benzoic acid and others well known in the art. Statine- like isosteres, hydroxyethylene isosteres, reduced amide bond isosteres, thioamide isosteres, urea isosteres, carbamate isosteres, thioether isosteres, vinyl isosteres and other amide bond isosteres known to the art may also be used. The use of analogues or non-conventional amino acids may improve the stability and biological half-life of the added peptide since they are more resistant to breakdown under physiological conditions. The person skilled in the art will be aware of similar types of substitution which may be made. A non-limiting list of non- conventional amino acids which may be used as suitable building blocks for a peptide and their standard abbreviations (in brackets) is as follows: a-aminobutyric acid (Abu), L-N- methylalanine (Nmala), a-amino-a-methylbutyrate (Mgabu), L-N-methylarginine (Nmarg), aminocyclopropane (Cpro), L-N-methylasparagine (Nmasn), carboxylate L-N-methylaspartic acid (Nmasp), aniinoisobutyric acid (Aib), L-N-methylcysteine (Nmcys), aminonorbomyl (Norb), L-N-methylglutamine (Nmgln), carboxylate L-N-methylglutamic acid (Nmglu), cyclohexylalanine (Chexa), L-N-methylhistidine (Nmhis), cyclopentylalanine (Cpen), L-N- methylisolleucine (Nmile), L-N-methylleucine (Nmleu), L-N-methyllysine (Nmlys), L-N- methylmethionine (Nmmet), L-N-methylnorleucine (Nmnle), L-N-methylnorvaline (Nmnva), L-N-methylomithine (Nmom), L-N-methylphenylalanine (Nmphe), L-N-methylproline (Nmpro), L-N-methylserine (Nmser), L-N-methylthreonine (Nmthr), L-N-methyltryptophan (Nmtrp), D-omithine (Dorn), L-N-methyltyrosine (Nmtyr), L-N-methylvaline (Nmval), L-N- methylethylglycine (Nmetg), L-N-methyl-t-butylglycine (Nmtbug), L-norleucine (NIe), L- norvaline (Nva), a-methyl-aminoisobutyrate (Maib), a-mcthyl-y-aminobutyratc (Mgabu), D-a- methylalanine (Dmala), a-methylcyclohexylalanine (Mchexa), D-a-methylarginine (Dmarg), a-methylcylcopentylalanine (Mcpen), D-a-methylasparagine (Dmasn), a-methyl-a- napthylalanine (Manap), D-a-methylaspartate (Dmasp), a-methylpenicillamine (Mpen), D-a- methylcysteine (Dmcys), N-(4-aminobutyl)glycine (Nglu), D-a-methylglutamine (Dmgln), N- (2-aminoethyl)glycine (Naeg), D-a-methylhistidine (Dmhis), N-(3 -aminopropyl)glycine (Norn), D-a-methylisoleucine (Dmile), N-amino-a-methylbutyrate (Nmaabu), D-a- methylleucine (Dmleu), a-napthylalanine (Anap), D-a-methyllysine (Dmlys), N-benzylglycine (Nphe), D-a-methylmethionine (Dmmet), N-(2-carbamylethyl)glycine (Ngln), D-a- methylomithine (Dmom), N-(carbamylmethyl)glycine (Nasn), D-a-methylphenylalanine (Dmphe), N-(2-carboxyethyl)glycine (Nglu), D-a-methylproline (Dmpro), N- (carboxymethyl)glycine (Nasp), D-a-methylserine (Dmser), N-cyclobutylglycine (Ncbut), D- a-methylthreonine (Dmthr), N-cycloheptylglycine (Nchep), D-a-methyltryptophan (Dmtrp), N-cyclohexylglycine (Nchex), D-a-methyltyrosine (Dmty), N-cyclodecylglycine (Ncdec), D- a-methylvaline (Dmval), N-cylcododecylglycine (Ncdod), D-N-methylalanine (Dnmala), N- cyclooctylglycine (Ncoct), D-N-methylarginine (Dnmarg), N-cyclopropylglycine (Ncpro), D- N-methylasparagine (Dnmasn), N-cycloundecylglycine (Ncund), D-N-methylaspartate (Dnmasp), N-(2,2-diphenylethyl)glycine (Nbhm), D-N-methylcysteine (Dnmcys), N-(3,3- diphenylpropyl)glycine (Nbhe), D-N-methylglutamine (Dnmgln), N-(3 guanidinopropyl)glycine (Narg), D-N-methylglutamate (Dnmglu), N-( 1 hydroxyethyl)glycine (Ntbx), D-N-methylhistidine (Dnmhis), N-(hydroxyethyl))glycine (Nser), D-N-methylisoleucine (Dnmile), N-(imidazolylethyl))glycine (Nhis), D-N- methylleucine (Dnmleu), N-(3 -indolylyethyl)glycine (Nhtrp), D-N-methyllysine (Dnnilys), N- mcthyl-y-aminobutyratc (Nmgabu), N-methylcyclohexylalanine (Nmchexa), D-N- methylmethionine (Dnmmet), D-N-methylomithine (Dnmom), N-methylcyclopentylalanine (Nmcpen), N-methylglycine (Nala), D-N-methylphenylalanine (Dnmphe), N- methylaminoisobutyrate (Nmaib), D-N-methylproline (Dnmpro), N-( 1 -methylpropyl)glycine (Nile), D-N-methylserine (Dnmser), N-(2-methylpropyl)glycine (Nleu), D-N-methylthreonine (Dnmthr), D-N-methyltryptophan (Dnmtrp), N-(l-methylethyl)glycine (Nval), D-N- methyltyrosine (Dnmtyr), N-methyla-napthylalanine (Nmanap), D-N-methylvaline (Dnmval), N-methylpenicillamine (Nmpen), g-aminobutyric acid (Gabu), N-(p-hydroxyphenyl)glycine (Nhtyr), L-/-butylglycine (Tbug), N-(thiomethyl)glycine (Ncys), L-ethylglycine (Etg), penicillamine (Pen), L-homophenylalanine (Hphe), L-a-methylalanine (Mala), L-a- methylarginine (Marg), L-a-methylasparagine (Masn), L-a-methylaspartate (Masp), L-a- methyl-t-butylglycine (Mtbug), L-a-methylcysteine (Mcys), L-methylethylglycine (Metg), L- a-methylglutamine (Mgln), L-a-methylglutamate (Mglu), L-a-methylhistidine (Mhis), L-a- methylhomophenylalanine (Mhphe), L-a-methylisoleucine (Mile), N-(2- methylthioethyl)glycine (Nmet), L-a-methylleucine (Mleu), L-a-methyllysine (Mlys), L-a- methylmethionine (Mmet), L-a-methylnorleucine (Mnle), L-a-methylnorvaline (Mnva), L-a- methylomithine (Mom), L-a-methylphenylalanine (Mphe), L-a-methylproline (Mpro), L-a- methylserine (Mser), L-a-methylthreonine (Mthr), L-a-methyltryptophan (Mtrp), L-a- methyltyrosine (Mtyr), L-a-methylvaline (Mval), L-N-methylhomophenylalanine (Nmhphe), N-(N-(2,2-diphenylethyl)carbamylmethyl)glycine (Nnbhm), N-(N-(3 ,3 -diphenylpropyl)- carbamylmethyl)glycine (Nnbhe), 1 -carboxy- 1 -(2,2-diphenyl-ethylamino)cyclopropane

(Nmbc), L-O-methyl serine (Omser), L-O-methyl homoserine (Omhser).

The term“cytotoxic effect” refers to the depletion, elimination and/or the killing of a target cell(s). The term“cyctotoxic agent” as used herein refers to an agent that has a cytotoxic and/or cytostatic effect on a cell. The term is intended to include chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, and fragments thereof. The term“cytostatic effect” refers to the inhibition of cell proliferation. The term “cytostatic agent” refers to an agent that has a cytostatic effect on a cell, thereby inhibiting the growth and/or expansion of a specific subset of cells.

The term“cytokine” as used herein refers to small proteins (~5-20 kDa) that are involved in autocrine signalling, paracrine signalling and endocrine signalling as immunomodulating agents. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumour necrosis factors but generally not hormones or growth factors.

The term“immunomodulatory molecule” as used herein refers to substance that stimulates or suppresses the immune system and may help the body fight cancer, infection, or other diseases. Specific immunomodulating molecules can be monoclonal antibodies, cytokines, and vaccines, which affect specific parts of the immune system.

The term“amphiphilic substance” as used herein refers to compounds with both hydrophilic and lipophilic properties. Common amphiphilic substances are phospholipids, cholesterol, glycolipids, fatty acids, bile acids, saponins, pepducins, local anaesthetics, Ab proteins and antimicrobial peptides.

The terms "protein" and "polypeptide" are used interchangeably herein and refer to any peptide -bond-linked chain of amino acids, regardless of length or post-translational modification. Preferably, the amino acid is any of the above-defined amino acids. Proteins usable in the present invention (including protein derivatives, protein variants, protein fragments, protein segments, protein epitopes and protein domains) can be further modified by chemical modification. This means such a chemically modified polypeptide comprises other chemical groups than the 20 naturally occurring amino acids. Examples of such other chemical groups include without limitation glycosylated amino acids and phosphorylated amino acids. Chemical modifications of a polypeptide may provide advantageous properties as compared to the parent polypeptide, e.g. one or more of enhanced stability, increased biological half-life, or increased water solubility.

The term“N-containing aromatic or non-aromatic mono or bicyclic heterocycle” as used herein refers to a cyclic saturated or unsaturated hydrocarbon compound which contains at least one nitrogen atom as constituent of the cyclic chain.

The term“nucleic acid” and the term“polynucleotide” are used interchangeably herein and refer to polymeric or oligomeric macromolecules, or large biological molecules, essential for all known forms of life. Nucleic acids, which include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are made from monomers known as nucleotides. Most naturally occurring DNA molecules consist of two complementary biopolymer strands coiled around each other to form a double helix. The DNA strand is also known as polynucleotides consisting of nucleotides. Each nucleotide is composed of a nitrogen-containing nucleobase as well as a monosaccharide sugar called deoxyribose or ribose and a phosphate group. Naturally occurring nucleobases comprise guanine (G), adenine (A), thymine (T), uracil (U) or cytosine (C). The nucleotides are joined to one another in a chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. If the sugar is desoxyribose, the polymer is DNA. If the sugar is ribose, the polymer is RNA. Typically, a polynucleotide is formed through phosphodiester bonds between the individual nucleotide monomers. In the context of the present invention the term“nucleic acid” includes but is not limited to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof such as e.g. RNA-DNA hybrids (within one strand), as well as cDNA, genomic DNA, recombinant DNA, cRNA and mRNA. A nucleic acid may consist of an entire gene, or a portion thereof, the nucleic acid may also be a miRNA, siRNA, piRNA or shRNA. MiRNAs are short ribonucleic acid (RNA) molecules, which are on average 22 nucleotides long but may be longer and which are found in all eukaryotic cells, i.e. in plants, animals, and some viruses, which functions in transcriptional and post-transcriptional regulation of gene expression. MiRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression and gene silencing. Small interfering RNAs (siRNAs), sometimes known as short interfering RNA or silencing RNA, are short ribonucleic acid (RNA molecules), between 20 - 25 nucleotides in length. They are involved in the RNA interference (RNAi) pathway, where they interfere with the expression of specific genes. A short hairpin RNA (shRNA) or small hairpin RNA (shRNA) is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. PiRNAs are also short RNAs which usually comprise 26 - 31 nucleotides and derive their name from so-called piwi proteins they are binding to. The nucleic acid can also be an artificial nucleic acid. Artificial nucleic acids include polyamide or peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally-occurring DNA or RNA by changes to the backbone of the molecule. The nucleic acids, can e.g. be synthesized chemically, e.g. in accordance with the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584).

The term“viral structural protein” (VSP) as used herein refers to viral coat proteins (VCP) or viral envelope glycoproteins (VEG). The term“viral coat protein” (VCP) as used in the context of the present invention refers to a structural virus capsid protein of a virus. Preferably the virus is a double-stranded DNA virus, single-stranded DNA virus, double-stranded RNA virus, single-stranded RNA virus, negative-sense single-stranded RNA virus, single-stranded RNA reverse transcribing virus, double-stranded RNA reverse transcribing virus. The VCP can comprise major capsid proteins of adeno-associated virus (AAV).

The term“viral envelope glycoproteins” (VEG) is used in the context of the present invention to refer to viral proteins that are part of the viral envelope. The viral envelope is typically derived from portions of the host cell membrane, e.g. comprises phospholipids, and additionally comprise viral glycoproteins that, e.g. help the virus to avoid the immune system. Enveloped viruses comprise DNA viruses, in particular Herpesviruses, Poxviruses, and Hepadnaviruses; RNA viruses, in particular Flavivirus, Togavirus, Coronavirus, Hepatitis D, Orthomyxovirus, Paramyxovirus, Rhabdovirus, Bunyavirus, Filovirus and Retroviruses. Accordingly, the viral envelop glycoprotein is preferably derived from any of these viruses.

The term "pharmaceutically acceptable salt" refers to a salt of the compound of the present invention. Suitable pharmaceutically acceptable salts of the compound of the present invention include acid addition salts which may, for example, be formed by mixing a solution of choline or derivative thereof with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the compound of the invention carries an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium and amine cations formed using counteranions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate). Illustrative examples of pharmaceutically acceptable salts include but are not limited to: acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate, valerate, and the like (see, for example, Berge, S. M., et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compounds which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide a compound of formula (I). A prodrug is an active or inactive compound that is modified chemically through in vivo physiological action, such as hydrolysis, metabolism and the like, into a compound of this invention following administration of the prodrug to a patient. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme. The suitability and techniques involved in making and using prodrugs are well known by those skilled in the art. For a general discussion of prodrugs involving esters see Svensson and Tunek Drug Metabolism Reviews 16.5 (1988) and Bundgaard Design of Prodrugs, Elsevier (1985). Examples of a masked carboxylate anion include a variety of esters, such as alkyl (for example, methyl, ethyl), cycloalkyl (for example, cyclohexyl), aralkyl (for example, benzyl, p- methoxybenzyl), and alkylcarbonyloxyalkyl (for example, pivaloyloxymethyl). Amines have been masked as arylcarbonyloxymethyl substituted derivatives which are cleaved by esterases in vivo releasing the free drug and formaldehyde (Bungaard J. Med. Chem. 2503 (1989)). Also, drugs containing an acidic NH group, such as imidazole, imide, indole and the like, have been masked with N-acyloxymethyl groups (Bundgaard Design of Prodrugs, Elsevier (1985)). Hydroxyl groups have been masked as esters and ethers. EP 0 039 051 (Sloan and Little, Apr. 11, 1981) discloses Mannich-base hydroxamic acid prodrugs, their preparation and use.

Compounds according to the invention can be synthesized according to one or more of the following methods. It should be noted that the general procedures are shown as it relates to preparation of compounds having unspecified stereochemistry. However, such procedures are generally applicable to those compounds of a specific stereochemistry, e.g., where the stereochemistry about a group is (S) or (R). In addition, the compounds having one stereochemistry (e.g., (R)) can often be utilized to produce those having opposite stereochemistry (i.e., (S)) using well-known methods, for example, by inversion.

Certain compounds of the present invention can exist in unsolvated forms as well as in solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the present invention.

The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium ( ³ H), iodine-l25 ( ¹²⁵ I) or carbon-l4 ( ¹⁴ C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

The term“liposome” as used herein, refers to uni- or multilame llar (preferably 2, 3, 4, 5, 6, 7, 8, 9, and 10 lamellar) lipid structures enclosing an aqueous interior, depending on the number of lipid membranes formed. Lipids, which are capable of forming a liposomes include all substances having fatty or fat-like properties. Such lipids comprise an extended apolar residue (X) and usually a water soluble, polar, hydrophilic residue (Y), which can be characterized by the basic formula

X-Yn

Wherein n equals or is greater than zero. Lipids with n = 0 are termed“apolar lipids”, while lipids with n > 1 a referred to as“polar lipids”. Preferred lipids, which can make up the lipids in the liposomes of the present invention are selected from the group consisting of glycerides, glycerophospholipides, glycerophosphinolipids, glycerophosphonolipids, sulfolipids, sphingolipids, phospholipids, isoprenolides, steroids, stearines, steroles and carbohydrate containing lipids.

A virus like particle (VLP) is a multimer of VSP, preferably of VCPs and/or VEPs that does not comprise polynucleotides but which otherwise has properties of a virus, e.g. binds to cell surface receptors, is internalized with the receptor, is stable in blood, and/or comprises glycoproteins etc.. VLPs are typically assembled of multimers of VCPs and/or VEPs, in particular of VCPs. VLPs are well known in the art and have been produced from a number of viruses including Parvoviridae (e.g. adeno-associated virus), Retroviridae (e.g. HIV), Flaviviridae (e.g. Hepatitis C virus) and bacteriophages (e.g. QP, AP205).

The term“pharmaceutical composition” as used herein refers to a substance and/or a combination of substances being used for the identification, prevention or treatment of a tissue status or disease. The pharmaceutical composition is formulated to be suitable for administration to a patient in order to prevent and/or treat disease. Further a pharmaceutical composition refers to the combination of an active agent with a carrier, inert or active, making the composition suitable for therapeutic use. Pharmaceutical compositions can be formulated for oral, parenteral, topical, inhalative, rectal, sublingual, transdermal, subcutaneous or vaginal application routes according to their chemical and physical properties. Pharmaceutical compositions comprise solid, semisolid, liquid, transdermal therapeutic systems (TTS). Solid compositions are selected from the group consisting of tablets, coated tablets, powder, granulate, pellets, capsules, effervescent tablets or transdermal therapeutic systems. Also comprised are liquid compositions, selected from the group consisting of solutions, syrups, infusions, extracts, solutions for intravenous application, solutions for infusion or solutions of the carrier systems of the present invention. Semisolid compositions that can be used in the context of the invention comprise emulsion, suspension, creams, lotions, gels, globules, buccal tablets and suppositories.

“Pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term“carrier”, as used herein, refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

The term“fibroblast activation protein (FAP)” as used herein is also known under the term“seprase”. Both terms can be used interchangeably herein. Fibroblast activation protein is a homodimeric integral protein with dipeptidyl peptidase IV (DPPIV)-like fold, featuring an alpha/beta-hydrolase domain and an eight-bladed beta-propeller domain.

Embodiments

In the following different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

In a first aspect, the present invention provides a compound of Formula (I)

wherein

Q, R, U, V, W, Y, Z are individually present or absent under the proviso that at least three of Q, R, U, V, W, Y, Z are present;

Q, R, U, V, W, Y, Z are independently selected form the group consisting of O, CH ₂ , NR ⁴ , C=0, C=S, C=NR ⁴ , HCR ⁴ and R ⁴ CR ⁴ , with the proviso that two Os are not directly adjacent to each other; preferably out of the six four groups are present of which two are C=0, one is CH ₂ and one is NH; more preferably four groups are present of which two are C=0, one is CH ₂ and one is NH; most preferably, V, W, Y and Z are present of which V and Z are C=0 and W and Y are independently selected from CH ₂ and NH;

R ¹ and R ² are independently selected from the group consisting of -H, -OH, halo, Ci-6-alkyl, - O-Ci-6-alkyl, S-Ci-e-alkyl; R ³ is selected from the group consisting of -H , -CN , -B(OH) ₂ , -C(O) -alkyl, -C(O) -aryl-, - C=C-C(0) -aryl, -C=C-S(0) ₂ -aryl, -C0 ₂ H , -SOsH , -S0 ₂ NH ₂ ,-P0 ₃ H ₂ , and 5-tetrazolyl;

R ⁴ is selected from the group consisting of -H, -Ci-6-alkyl, -O-Ci-6-alkyl, -S-Ci-6-alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl, and -Ci-6-aralkyl, each of said - Ci-6-alkyl being optionally substituted with from 1 to 3 substituents selected from -OH, oxo, halo and optionally connected to Q, R, U, V, W, Y or Z;

R ⁵ is selected from the group consisting of -H, halo and Ci-6-alkyl;

R ⁶ , and R ⁷ are independently selected from the group consisting of-H, . under the proviso that R ⁶ and R ⁷ are not at the same time H, preferably R ⁶ is attached to the 7- or 8-quinolyl position and R ⁷ is attached to the 5- or 6- quinolyl position; more preferably R ⁶ is attached to the 7-quinolyl position and R ⁷ is attached to the 6-quinolyl position,

wherein L is a linker,

wherein D, A, E, and B are individually present or absent, preferably wherein at least A, E, and B are present, wherein when present:

D is a linker;

A is selected from the group consisting of NR ⁴ , O, S, and CEb;

E is selected from the group consisting of

wherein i is 1, 2, or 3;

wherein j is 1, 2, or 3;

wherein k is 1, 2, or 3;

wherein m is 1, 2, or 3;

more preferably, E is Ci-6-alkyl, most preferably, E is C3 or C4 alkyl;

B is selected from the group consisting of S, NR ⁴ , NR ⁴ -0, NR ⁴ -Ci-6-alkyl, NR ⁴ -Ci-6-alkyl-NR ⁴ , and a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein NR ⁴ -Ci-6-alkyl-NR ⁴ and the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of Ci-6-alkyl, aryl, Ci-6-aralkyl; and

R ⁸ is selected from the group consisting of a cytostatic and/or cytotoxic agent, a cytokine, an immunomodulatory molecule, an amphiphilic substance, polyglycolic acid, polylactic acid or a derivative thereof a nucleic acid, a viral structural protein, a protein, biotin and combinations thereof; is a l-naphtyl moiety or a 5 to 10- membered N-containing aromatic or non- aromatic mono- or bicyclic heterocycle, wherein there are 2 ring atoms between the N atom and X; said heterocycle optionally further comprising 1, 2 or 3 heteroatoms selected from O, N and S; and X is a C atom;

or a pharmaceutically acceptable tautomer, racemate, hydrate, solvate, or salt thereof. Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a preferred embodiment of the first aspect of the present invention,

Q, R, U are CH ₂ and are individually present or absent; preferably, Q and R are absent;

V is CH ₂ , C=0, C=S or C=NR ⁴ ; preferably, V is C=0;

W is NR ⁴ ; preferably, W is NH;

Y is HCR ⁴ ; preferably, Y is CH ₂ ; and

Z is C=0, C=S or C=NR ⁴ , preferably, Z is C=0.

In a further preferred embodiment of the first aspect of the present invention,

Q, R, U are absent;

V is CH ₂ ;

W is NH;

Y is CH ₂ ; and

Z is C=0.

In a further preferred embodiment of the first aspect of the present invention,

R ¹ and R ² are independently selected from the group consisting of -H and halo; preferably, R ¹ and R ² are halo; more preferably, R ¹ and R ² are F;

R ³ is selected from the group consisting of -H, -CN, and -B(OH) ₂ ; preferably, R ³ is -CN or - B(OH) ₂ ; more preferably, R ³ is -CN; R ⁴ is selected from the group consisting of -H and -Ci-6-alkyl, wherein the -Ci-6-alkyl is optionally substituted with from 1 to 3 substituents selected from -OH. Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert- butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

Q, R, U are absent;

V is CH ₂ ;

W is NH;

Y is CH ₂ ;

Z is C=0;

R ¹ and R ² are independently selected from the group consisting of -H and halo; preferably, R ¹ and R ² are halo; more preferably, R ¹ and R ² are F;

R ³ is selected from the group consisting of -H, -CN, and -B(OH) ₂ ; preferably, R ³ is -CN or - B(OH) ₂ ; more preferably, R ³ is -CN;

R ⁴ is selected from the group consisting of -H and -Ci-6-alkyl, wherein the -Ci-6-alkyl is optionally substituted with from 1 to 3 substituents selected from -OH. Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert- butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

Q, R, U are absent;

V is CH ₂ ;

W is CH ₂ ;

Y is NH;

Z is C=0;

R ¹ and R ² are independently selected from the group consisting of -H and halo; preferably, R ¹ and R ² are halo; more preferably, R ¹ and R ² are F;

R ³ is selected from the group consisting of -H, -CN, and -B(OH) ₂ ; preferably, R ³ is -CN or - B(OH) ₂ ; more preferably, R ³ is -CN;

R ⁴ is selected from the group consisting of -H and -Ci-6-alkyl, wherein the -Ci-6-alkyl is optionally substituted with from 1 to 3 substituents selected from -OH. Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert- butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention, is selected from the group consisting

optionally further comprising 1 or 2 heteroatoms selected from O, N, and S.

In a further preferred embodiment of the first aspect of the present invention,

, optionally ffurther comprising 1 or 2 heteroatoms selected from O, N, and S.

In a further preferred embodiment of the first aspect of the present invention, is selected from the group consisting of

In a preferred embodiment, is selected from the group consisting of

In another preferred embodiment

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R ⁷ is , preferably R ⁷ is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein

D is absent or is present and is a cleavable linker such as a peptide motif that is cleaved by cathepsin;

A is O;

E is Ci- ₆ -alkyl or , wherein m is 1, 2, or 3; Preferably, Ci- ₆ -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is Ci- ₆ -alkyl, most preferably, E is C3 or C4 alkyl;

B is NR ⁴ -Ci- ₆ -alkyl or a 5- to lO-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of Ci- ₆ -alkyl, aryl, Ci-6-aralkyl. Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R ⁷ is , preferably R ⁷ is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein

D is absent or is present and is a cleavable linker such as a peptide motif that is cleaved by cathepsin;

A is S;

E is Ci-6-alkyl or , wherein m is 1, 2, or 3; Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is Ci-6-alkyl, most preferably, E is C3 or C4 alkyl;

B is NR ⁴ -Ci-6-alkyl or a 5- to lO-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of Ci-6-alkyl, aryl, Ci-6-aralkyl. Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R ⁷ is , preferably R ⁷ is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein

D is absent or is present and is a cleavable linker such as a peptide motif that is cleaved by cathepsin;

A is CEE;

E is Ci-6-alkyl or , wherein m is 1, 2, or 3; Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is Ci-6-alkyl, most preferably, E is C3 or C4 alkyl; B is NR ⁴ -Ci-6-alkyl or a 5- to lO-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of Ci-6-alkyl, aryl, Ci-6-aralkyl. Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R 8 rv

D^ 'Έ' V _

R is , preferably R is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein

D is absent or is present and is a cleavable linker such as a peptide motif that is cleaved by cathepsin;

A is NH;

E is Ci-6-alkyl or , wherein m is 1, 2, or 3; Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is Ci-6-alkyl, most preferably, E is C3 or C4 alkyl;

B is NR ⁴ -Ci-6-alkyl or a 5- to lO-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of Ci-6-alkyl, aryl, Ci-6-aralkyl. Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R ⁷ is , preferably R ⁷ is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein

D is an amino acid, preferably carrying a charged side chain;

A is O;

E is Ci-6-alkyl or , wherein m is 1, 2, or 3; Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is Ci-6-alkyl, most preferably, E is C3 or C4 alkyl;

B is NR ⁴ -Ci-6-alkyl or a 5- to lO-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of Ci-6-alkyl, aryl, Ci-6-aralkyl. Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R ⁷ is , preferably R ⁷ is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein

D is an amino acid, preferably carrying a charged side chain;

A is S;

E is Ci-6-alkyl or , wherein m is 1, 2, or 3; Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is Ci-6-alkyl, most preferably, E is C3 or C4 alkyl;

B is NR ⁴ -Ci-6-alkyl or a 5- to lO-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of Ci-6-alkyl, aryl, Ci-6-aralkyl. Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H; ₇

R is , preferably R is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein

D is an amino acid, preferably carrying a charged side chain; A is CEE;

E is Ci-6-alkyl or , wherein m is 1, 2, or 3; Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is Ci-6-alkyl, most preferably, E is C3 or C4 alkyl;

B is NR ⁴ -Ci-6-alkyl or a 5- to lO-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of Ci-6-alkyl, aryl, Ci-6-aralkyl. Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R is , preferably R is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein

D is an amino acid, preferably carrying a charged side chain;

A is NH;

E is Ci-6-alkyl or , wherein m is 1, 2, or 3; Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is Ci-6-alkyl, most preferably, E is C3 or C4 alkyl;

B is NR ⁴ -Ci-6-alkyl or a 5- to lO-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of Ci-6-alkyl, aryl, Ci-6-aralkyl. Preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R ⁷ is , preferably R ⁷ is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein D is absent or is present and is a cleavable linker such as a peptide motif that is cleaved by cathepsin;

A is O;

E is Ci-6-alkyl or , wherein m is 1, 2, or 3; Preferably, E is C 1-6- alkyl and Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is Ci-6-alkyl, most preferably, E is C3 or C4 alkyl;

B is a 5- to lO-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 nitrogen atoms.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R is , preferably R is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein

D is absent or is present and is a cleavable linker such as a peptide motif that is cleaved by cathepsin;

A is O;

E is C3 or C4 alkyl; more preferably, E is propyl or butyl;

B is a 5- to lO-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 nitrogen atoms.

In a further preferred embodiment of the first aspect of the present invention, the N-containing heterocycle comprised in B is an aromatic or non-aromatic monocyclic heterocycle:

, wherein

the heterocycle optionally further comprises 1 or 2 heteroatoms selected form O, N and S, optionally further comprises 1 nitrogen;

—I is attached to position 1, 2, or 3, preferably to position 2;

1 is 1 or 2.

In a further preferred embodiment of the first aspect of the present invention, the N-containing heterocycle comprised in B is selected from the group consisting of:

wherein if the N-containing heterocycle comprised in B is ,

the heterocycle optionally further comprises 1 or 2 heteroatoms selected from O, N and S, optionally further comprises 1 nitrogen, optionally compromises one or more (e.g. amino acid derived) side chains;

—I ¹ is attached to position 1, 2, or 3, preferably to position 2;

o is 1 or 2;

preferably, if the N-containing heterocycle comprised in B is , the N-

containing heterocycle comprised in B is selected from the group consisting of

In a further preferred embodiment of the first aspect of the present invention, the N-containing heterocycle comprised in B is selected from the group consisting of:

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R ⁷ is . preferably R ⁷ is attached to the 6-quinolyl position, wherein

D is absent or is present and is a cleavable linker such as a peptide motif that is cleaved by cathepsin;

A is O;

E is propyl or butyl;

In a further preferred embodiment of the first aspect of the present invention,

Q, R, U are absent;

V is C=0;

W is NH;

Y is CH ₂ ;

Z is C=0;

R ¹ and R ² are independently selected from the group consisting of -H and halo; preferably, R ¹ and R ² are independently selected from the group consisting of -H and F; more preferably, R ¹ and R ² are the same and are selected from the group consisting of -H and F;

R ³ is -CN;

R ⁵ and R ⁶ are H;

R ⁷ is . preferably R ⁷ is attached to the 6-quinolyl position, wherein

D is absent or is present and is a cleavable linker such as a peptide motif that is cleaved by cathepsin;

A is O;

E is Ci- ₆ -alkyl or , wherein m is 1, 2, or 3; preferably, E is Ci- ₆ - alkyl; preferably, Ci- ₆ -alkyl is selected from the group consisting of methyl, ethyl, propyl, i- propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is Ci-6-alkyl, most preferably, E is C3 or C4 alkyl;

B is NH-Ci-e-alkyl,

preferably, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl,

butyl, sec-butyl, tert-butyl, pentyl and hexyl; preferably, B i ; and

In a further preferred embodiment of the first aspect of the present invention,

Q, R, U are absent;

V is C=0;

W is NH;

Y is CEE;

Z is C=0;

R ¹ and R ² are the same and are selected from the group consisting of -H and F;

R ³ is -CN;

R ⁵ and R ⁶ are H;

R ⁷ is , preferably R ⁷ is attached to the 6-quinolyl position, wherein

D is absent or is present and is a cleavable linker such as a peptide motif that is cleaved by cathepsin;

A is O;

E is methyl, ethyl, propyl or butyl;

In a further preferred embodiment of the first aspect of the present invention,

Q, R, U are absent;

V is C=0;

W is NH;

Y is CH ₂ ;

Z is C=0;

R ¹ and R ² are the same and are selected from the group consisting of -H and F;

R ³ is -CN;

R ⁵ and R ⁶ are H;

R is , R is attached to the 6-quinolyl position, wherein

D is absent or is present and is a cleavable linker such as a peptide motif that is cleaved by cathepsin;

A is O;

E is methyl, ethyl, propyl or butyl;

In a further preferred embodiment of the first aspect of the present invention, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert- butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention, Ci-6-aralkyl is selected from the group consisting of benzyl, phenyl-ethyl, phenyl-propyl, and phenyl-butyl. In a preferred embodiment of the first aspect of the present invention, the compound of the first aspect of the invention is selected from the compounds of table 1. More preferably, the compound of the first aspect of the invention is selected from the group consisting of FAPI-02 and F API-04.

Table 1: Preferred compounds of the first aspect of the invention.

Q, R, U are absent; R ⁵ is H; R ⁶ is attached to the 7-quinolyl position; R ⁷ is attached to the 6- quinolyl position; V is C=0; W is NH; Y is CFb; Z is C=0; R ³ is -CN; A is O, R ⁷ is attached to the 5-quinolyl position).

In a further preferred embodiment of the first aspect of the present invention, R ⁸ is a cytostatic and/or cytotoxic agent. Preferably, the cytostatic and/or cytotoxic agent is selected from the group consisting of alkylating substances, anti-metabolites, antibiotics, epothilones, nuclear receptor agonists and antagonists, anti-androgenes, anti-estrogens, platinum compounds, hormones and antihormones, interferons and inhibitors of cell cycle-dependent protein kinases (CDKs), inhibitors of cyclooxygenases and/or lipoxygenases, biogeneic fatty acids and fatty acid derivatives, including prostanoids and leukotrienes, inhibitors of protein kinases, inhibitors of protein phosphatases, inhibitors of lipid kinases, platinum coordination complexes, ethyleneimenes, methylmelamines, trazines, vinca alkaloids, pyrimidine analogs, purine analogs, alkylsulfonates, folic acid analogs, anthracendiones, substituted urea, methylhydrazin derivatives, in particular acediasulfone, aclarubicine, a-amanitin, ambazone, aminoglutethimide, L-asparaginase, monomethyl auristatin E, azathioprine, bleomycin, busulfan, calcium folinate, carboplatin, carpecitabine, carmustine, celecoxib, chlorambucil, cis- platin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin dapsone, daunorubicin, dibrompropamidine, diethylstilbestrole, docetaxel, dolastatin 10 and 15, doxorubicin, enediynes, epirubicin, epothilone B, epothilone D, estramucin phosphate, estrogen, ethinylestradiole, etoposide, flavopiridol, floxuridine, fludarabine, fluorouracil, fluoxymesterone, flutamide fosfestrol, furazolidone, gemcitabine, gonadotropin releasing hormone analog, hexamethylmelamine, hydroxycarbamide, hydroxymethylnitrofurantoin, hydroxyprogesteronecaproat, hydroxyurea, idarubicin, idoxuridine, ifosfamide, interferon a, irinotecan, leuprolide, lomustine, lurtotecan, mafenide sulfate olamide, mechlorethamine, medroxyprogesterone acetate, megastrolacetate, melphalan, mepacrine, mercaptopurine, methotrexate, metronidazole, mitomycin C, mitopodozide, mitotane, mitoxantrone, mithramycin, nalidixic acid, nifuratel, nifuroxazide, nifuralazine, nifurtimox, nimustine, ninorazole, nitrofurantoin, nitrogen mustards, oleomucin, oxolinic acid, pentamidine, pentostatin, phenazopyridine, phthalylsulfathiazole, pipobroman, prednimustine, prednisone, preussin, procarbazine, pyrimethamine, raltitrexed, rapamycin, rofecoxib, rosiglitazone, salazosulfapyridine, scriflavinium chloride, semustine streptozocine, sulfacarbamide, sulfacetamide, sulfachlopyridazine, sulfadiazine, sulfadicramide, sulfadimethoxine, sulfaethidole, sulfafurazole, sulfaguanidine, sulfaguanole, sulfamethizole, sulfamethoxazole, co-trimoxazole, sulfamethoxydiazine, sulfamethoxypyridazine, sulfamoxole, sulfanilamide, sulfaperin, sulfaphenazole, sulfathiazole, sulfisomidine, staurosporin, tamoxifen, taxol, teniposide, tertiposide, testolactone, testosteronpropionate, thioguanine, thiotepa, tinidazole, topotecan, triaziquone, treosulfan, trimethoprim, trofosfamide, UCN-01, vinblastine, vincristine, vindesine, vinblastine, vinorelbine, zorubicin, or their respective derivatives or analogs thereof and combinations thereof.

More preferably, the cytostatic and/or cytotoxic agent is selected from the group consisting of doxorubicin, a-amanitin and monomethyl auristatin E. In a particularly preferred embodiment, R ⁸ is doxorubicin.

In a further preferred embodiment of the first aspect of the present invention, R ⁸ is a cytokine. In one embodiment, the cytokine is a chemokine molecule. Preferably, the chemokine molecule is selected from the group consisting of CXCL9, CXCL10 and CX3CL1. In a particularly preferred embodiment, R ⁸ is CXCL9. In another particularly preferred embodiment, R ⁸ is CXCL10. In another particularly preferred embodiment, R ⁸ is CX3CL1.

In a further preferred embodiment of the first aspect of the present invention, R ⁸ is an immunomodulatory molecule. Preferably, the immunomodulatory molecule is selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CX3CL1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, interleukin-2, interferon alpha and interferon gamma. More preferably, the immunomodulatory molecule is selected from the group consisting of CXCL3, interleukin-2 and CCL8. In a particularly preferred embodiment, R ⁸ is interleukin-2.

In a further preferred embodiment of the first aspect of the present invention, R ⁸ is an amphiphilic substance. Preferably, the amphiphilic substance is selected from the group consisting of a lipid, a phospholipid and other highly lipophilic moiety conjugated to a polar group such as an ammonium ion or inositol triphosphate. In particular, the lipid is selected from the group consisting of saccharolipids, prenol lipids, sterol lipids, glycerolipids, polyketides and fatty acids and the phospholipid is selected from the group consisting of plasmalogens, sphingo lipids, phophatidates and phosphoinositides. More preferably, the amphiphilic substance is a lipid or a phospholipid. Most preferably, the amphiphilic substance is N- PEGylated l,2-disteaorylglycero-3-phosphoethanolamine. In a particular preferred embodiment R ⁸ is a lipid. In another particular preferred embodiment R ⁸ is a phospholipid. In another particular preferred embodiment R ⁸ is /V-PEGylatcd l,2-disteaorylglycero-3- phosphoethanolamine.

In a further preferred embodiment of the first aspect of the present invention, R ⁸ is a nucleic acid. Preferably, the nucleic acid is selected from the group consisting of DNA, RNA, siRNA, mRNA, PNA and cDNA. More preferably, the nucleic acid encodes a cytokine and/or an immunomodulatory molecule as defined above. More preferably, the nucleic acid is a siRNA or PNA.

In a further preferred embodiment of the first aspect of the present invention, R ⁸ is a viral structural protein. Preferably, the viral structural protein is of a virus selected from the group consisting of

(i) double-stranded DNA virus,

preferably Myoviridae, Siphoviridae, Podoviridae, Herpesviridae, Adenoviridae, Baculoviridae, Papillomaviridae, Polydnaviridae, Polyomaviridae, Poxviridae;

(ii) single-stranded DNA virus,

preferably Anelloviridae, Inoviridae, Parvoviridae;

(iii) double-stranded RNA virus,

preferably Reoviridae;

(iv) single-stranded RNA virus, preferably Coronaviridae, Picomaviridae, Caliciviridae, Togaviridae, Flaviviridae, Astroviridae, Arteriviridae, Hepeviridae;

(v) negative-sense single-stranded RNA virus,

preferably Arenaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Bunyaviridae, Orthomyxoviridae, Bomaviridae;

(vi) single-stranded RNA reverse transcribing virus,

preferably Retroviridae;

(vii) double-stranded DNA reverse transcribing virus,

preferably Caulimoviridae, Hepadnaviridae.

More preferably, the viral structural protein, preferably VCP is derived from a virus selected from the group consisting of double-stranded DNA virus, preferably Myoviridae, Siphoviridae, Podoviridae, Herpesviridae, Adenoviridae, Baculoviridae, Papillomaviridae, Polydnaviridae, Polyomaviridae, Poxviridae; single-stranded DNA virus, preferably Anelloviridae, Inoviridae, Parvoviridae; double-stranded RNA virus, preferably Reoviridae; single-stranded RNA virus, preferably Coronaviridae, Picomaviridae, Caliciviridae, Togaviridae, Flaviviridae, Astroviridae, Arteriviridae, Hepeviridae; negative-sense single- stranded RNA vims, preferably Arenaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Bunyaviridae, Orthomyxoviridae, Bomaviridae; single-stranded RNA reverse transcribing vims, preferably Retroviridae; double-stranded DNA reverse transcribing vims, preferably Caulimoviridae, Hepadnaviridae. More preferably, the VCP is from a family of the Parvoviridae, preferably from adeno-associated vims. Even more preferably, the AAV is human AAV, bovine AAV, caprine AAV, avian AAV, canine parvovirus (CPV), mouse parvovirus; minute vims of mice (MVM); parvovirus B19 (B19); parvovirus Hl (Hl); human bocavims (HBoV); feline panleukopenia vims (FPV); or goose parvovirus (GPV). Even more preferably, the VCP is from a certain AAV-serotype, preferably AAV-l, AAV-2, AAV-2- AAV-3 hybrid, AAV-3a, AAV-3b, AAV-4, AAV-5, AAV-6, AAV-6.2, AAV-7, AAV-8, AAV-9, AAV-10, AAVrh.lO, AAV-l 1, AAV-12, AAV-13 or AAVrh32.33. More preferably, the VCP is from AAV-2 or a variant thereof that is capable of assembling into a VLP.

In a further preferred embodiment of the first aspect of the present invention, R ⁸ is protein, wherein the protein is selected from the group consisting of a membrane bound protein and unbound protein. Examples of the protein include but are not limited to CEA, CA19-9, Macrophage Migration Inhibition Factor (MIF), IL-8 (interleukin 8), AXL, MER and c-MET.

In a further preferred embodiment of the first aspect of the present invention, R ⁸ is biotin. In a second aspect, the present invention relates to a liposome comprising or consisting of the compound of the first aspect, wherein R ⁸ is an amphiphilic substance.

The liposomes of the present invention can be various types of liposomes, for example, as described in Alavi et ah, Adv Pharm Bull, 2017. In one embodiment, the liposomes of the present invention is a stealth liposome. Stealth liposomes are well known in the art and are for example reviewed by Immordino et al., Int J Nanomedicine, 2006.

The liposome of the present invention can be positively charged, negatively charged or neutral liposomes. The charge of a liposome is determined by the lipid composition and is the average of all charges of the lipids comprised in the liposome. For example, a mixture of a negatively charged phospholipid and cholesterol will yield a negatively charged liposome.

Preferred lipids/phospholipids to be used in liposomes include but are not limited to glycerides, glycerophospholipides, glycerophosphinolipids, glycerophosphonolipids, sulfolipids, sphingolipids, phospholipids, isoprenolides, steroids, stearines, steroles and carbohydrate containing lipids.

In a preferred embodiment, the negatively charged lipid/phospholipid is selected from the group consisting of phosphatidylserine (PS), phosphatidylglycerol (PG) and phosphatidic acid (PA). PS and PG are collective terms for lipids sharing a similar phosphatidylserine and phosphatidylglycerol, respectively, head group. However, many different apolar residues can be attached to these head groups. Thus, PSs and PGs isolated from different natural sources vary substantially in the length, composition and/or chemical structure of the attached apolar residues and naturally occurring PS and PG usually is a mixture of PSs and PGs with different apolar residues.

The PS employed in the liposomes of the present invention is preferably selected from the group consisting of palmitoyloleoylphosphatidylserine, palmitoyllinoeoyl- phosphatidylserine, palmitoylarachidonoylphosphatidylserine, palmitoyldocosahexaenoyl- phosphatidylserine, stearoyloleoylphosphatidylserine, stearoyllinoleoylphosphatidylserine, stearoyl-arachidonoylphosphatidylserine, stearoyldocosahexaenoylphosphatidylserine, dicaprylphosphatidylserine, dilauroylphosphatidylserine, dimyristoylphosphatidylserine, diphytanoylphosphatidylserine, diheptadecanoylphosphatidylserine, dioleoylphosphatidyl- serine, dipalmitoylphosphatidylserine, distearoylphosphatidylserine, dilinoleoy- lphosphatidylserine dierucoylphosphatidylserine, didocosahexaenoyl-phospahtidylserine, PS from brain, and PS from soy bean; particular preferred is dioleoylphosphatidylserine.

The PG employed in the liposome of the present invention is preferably selected from the group consisting of palmitoyloleoylphosphatidylglycerol, palmitoyl- linoleoylphosphatidylglycerol, palmitoylarachidonoylphosphatidylglycerol, palmitoyl- docosahexaenoylphosphatidylglycerol, stearoyloleoylphosphatidylglycerol, stearoyl- linoleoylphosphatidylglycerol, stearoylarachidonoylphosphatidylglycerol, stearoyldocosa- hexaenoylphosphatidylglycerol, dicaprylphosphatidylglycerol dilauroylphosphatidylglycerol, diheptadecanoylphosphatidylglycerol, diphytanoyl-phosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dielaidoylphosphatidyl- glycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dilinoeoyl- phosphatidylglycerol, diarachidonoylphosphatidylglycerol, docosahexaenoylphosphatidyl- glycerol, and PG from egg; in particular dioleoylphosphatidylglycerol.

Similar to PS and PG, PE is also a generic term for lipids sharing a phosphatidylethanolamine head group. Preferably, the PE is selected from the group consisting of palmitoyloleoylphosphatidylethanolamine, palmitoyllinoleoylphosphatidylethanolamine, palmitoylarachidonoylphosphatidylethanolamine, palmitoyldocosahexaenoylphosphatidyl- ethanolamine, stearoyloleoylphosphatidylethanolamine, stearoyllinoleoylphosphatidyl- ethanolamine, stearoylarachidonoylphosphatidylethanolamine, stearoyldocosahexaenoyl- phosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristoylphosphatidyl- ethanolamine, diphytanoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, diheptadecanoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine, diarachidonoylphosphatidylethanolamine, docosahexaenoyl- phosphatidylethanolamine, PE from bacteria, PE from heart, PE from brain, PE from liver, PE from egg, and PE from soybean., in particular l,2-diacyl-sn-glycero-3-PE, l-acyl-2-acyl-sn- glycero-3-PE, 1 ,2-dipalmitoyl-PE and/or l,2-dilauroyl-sn-glycero-3-PE (DLPE).

The liposome of the present invention can comprise at least one further component selected from the group consisting of an adjuvant, additive, and auxiliary substance. Preferably, adjuvants are selected from the group consisting of unmethylated DNA, in particular unmethylated DNA comprising CpG dinucleotides (CpG motif), in particular CpG ODN with phosphorothioate (PTO) backbone (CpG PTO ODN) or phosphodiester (PO) backbone (CpG PO ODN); bacterial products from the outer membrane of Gram-negative bacteria, in particular monophosphoryl lipid A (MPLA), lipopolysaccharides (LPS), muramyl dipeptides and derivatives thereof; synthetic lipopeptide derivatives, in particular Par Cys; lipoarabino- mannan; peptidoglycan; zymosan; heat shock proteins (HSP), in particular HSP 70; dsRNA and synthetic derivatives thereof, in particular Poly Epoly C; polycationic peptides, in particular poly-L-arginine; taxol; fibronectin; flagellin; imidazoquinoline; cytokines with adjuvant activity, in particular GM-CSF, interleukin- (IL-)2, IL-6, IL-7, IL-18, type I and II, interferons, in particular interferon-gamma, TNF-alpha; 25-dihydroxyvitamin D3 (calcitriol); synthetic oligopeptides, in particular MHCII-presented peptides; gel-like precipitates of aluminum hydroxide (alum). Particular preferred adjuvants, which can be comprised in the liposome of the present invention are selected from the group unmethylated DNA, in particular unmethylated DNA comprising CpG dinucleotides (CpG motif), in particular CpG ODN with phosphorothioate (PTO) backbone (CpG PTO ODN) or phosphodiester (PO) backbone (CpG PO ODN), bacterial products from the outer membrane of Gram-negative bacteria, in particular monophosphoryl lipid A (MPLA) and synthetic lipopeptide derivatives, in particular Par Cys.

The term“additive” comprises substances, which stabilize any component of the liposome or of the liquid medium like, for example, antioxidants, radical scavengers or the like. In particular, stabilizers are selected from the group consisting of a-tocopherol or carbohydrates, in particular glucose, sorbitol, sucrose, maltose, trehalose, lactose, cellubiose, raffmose, maltotriose, or dextran. The stabilizers can be comprised in the lipid membranes of the liposomes, the interior of the liposomes and/or within the liquid medium surrounding the liposomes.

Liposomes of the present invention can have a diameter between 10 and 1000 nm. They, however, have in a preferred embodiment a diameter of between 30 and 800 nm, preferably between 40 and 500 nm, preferably between 50 and 300 nm, and more preferably between 100 and 200 nm. The diameter of the liposomes can be affected, for example, by extrusion of the liposomal composition through sieves or meshes with a known pore size. This and further methods of controlling the size of liposomes are well known in the art and are described, for example, in Mayhew et al. (1984) Biochim. Biophys. Acta 775: 169-174 or Olson et al. (1979) Biochim. Biophys. Acta 557:9-23.

In a preferred embodiment of the second aspect of the invention, the liposome or the mixture of liposomes of the present invention are comprised in a liquid medium. The term “liquid medium” preferably comprises all biocompatible, physiological acceptable liquids and liquid compositions in particular FLO, aqueous salt solutions, and buffer solutions like, for example, PBS, Ringer solution and the like.

In a preferred embodiment of the second aspect of the present invention, the liposome is loaded with a substance selected from the group consisting of an agent and a nucleic acid.

The agent that the liposome is loaded with preferably is a cytostatic and/or cytotoxic agent as disclosed above. The nucleic acid that the liposome is loaded with preferably is a nucleic acid as disclosed above. A variety of methods are available in the prior art to "load" a liposome with a given therapeutic agent. In its simplest form the therapeutic agent(s) is(are) admixed with the lipid components during formation of the liposomes. Other passive loading methods include dehydration-rehydration (Kirby & Gregoriadis (1984) Biotechnology 2:979), reverse-phase evaporation (Szoka & Papahadjopoulos (1978) Proc. Natl.Acad. Sci. USA 75:4194), or detergent-depletion (Milsmann et al. (1978) Biochim. Biophys. Acta 512: 147-155). Other methodologies for encapsulating therapeutic agents include so called "remote loading" or "active loading" in which due to a gradient, for example, a pH or salt gradient between the exterior and the interior of a preformed liposome the therapeutic agent is transported into the liposome along the gradient (see, for example, Cheung et al. (1998) Biochim. Biophys. Acta 1414:205-216; Cullis et al. (1991) Trends Biotechnol. 9:268-272; Mayer et al. (1986) Chem. Phys. Lipids 40:333-345).

In a third aspect, the present invention relates to a virus-like particle (VLP) comprising or consisting of the compound of the first aspect of the present invention, wherein R ⁸ is a viral structural protein.

In a preferred embodiment of the third aspect of the present invention, the virus-like particle is loaded with a substance selected from the group consisting of an agent and a nucleic acid.

The agent that the virus-like particle is loaded with preferably is a cytostatic and/or cytotoxic agent as disclosed above. The nucleic acid that the virus-like particle is loaded with preferably is a nucleic acid as disclosed above.

In a fourth aspect, the present invention relates to a pharmaceutical composition comprising or consisting of at least one compound of the first aspect, or the liposome of the second aspect, or the virus-like particle of the third aspect, and, optionally, a pharmaceutically acceptable carrier and/or excipient.

In a fifth aspect, the present invention relates to the compound of the first aspect, or the liposome of the second aspect, or the virus-like particle of the third aspect, or the pharmaceutical composition of the fourth aspect for use in the treatment of a disease characterized by overexpression of fibroblast activation protein (FAP) in an animal or a human subject. Preferably, the disease characterized by overexpression of fibroblast activation protein (FAP) is selected from the group consisting of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and keloid disorder.

Preferably, if the disease characterized by overexpression of fibroblast activation protein (FAP) is cancer, the cancer is selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharynx cancer, nasopharynx cancer, larynx cancer, myeloma cells, bladder cancer, cholangiocellular carcinoma, clear cell renal carcinoma, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, CUP (carcinoma of unknown primary), thymus carcinoma, desmoid tumors, glioma, astrocytoma, cervix carcinoma and prostate cancer. Preferably, the cancer is breast cancer, colon cancer, lung cancer, head and neck cancer, liver cancer or pancreatic cancer. Even more preferably, the cancer is colon cancer.

Preferably, if the disease characterized by overexpression of fibroblast activation protein (FAP) is chronic inflammation, the chronic inflammation is selected from the group consisting of rheumatoid arthritis, osteoarthritis and Crohn’s disease. Preferably, the chronic inflammation is rheumatoid arthritis.

Preferably, if the disease characterized by overexpression of fibroblast activation protein (FAP) is fibrosis, the fibrosis is selected from the group consisting of pulmonary fibrosis, such as idiopathic pulmonary fibrosis and liver cirrhosis.

Preferably, if the disease characterized by overexpression of fibroblast activation protein (FAP) is tissue remodeling, the tissue remodeling occurs after myocardial infarction.

Preferably, if the disease characterized by overexpression of fibroblast activation protein (FAP) is a keloid disorder, the keloid disorder is selected from the group consisting of scar formation, keloid tumors and keloid scar.

In a sixth aspect, the present invention relates to a kit comprising or consisting of the compound of the first aspect, or the liposome of the second aspect, or the virus-like particle of the third aspect, or the pharmaceutical composition of the fourth aspect and instructions for the treatment of a disease. Preferably, the disease is a disease as specified above.

Examples

Example 1: Compound synthesis

Based on a FAP-a specific inhibitor (Jansen et ah, ACS Med Chem Lett, 2013) two radiotracers were synthesized. Radioiodine labeled FAPI-01 was obtained via an organotin stannylated precursor, which was prepared through palladium catalyzed bromine/tin exchange. FAPI-02 is a precursor for the chelation of radio metals which was synthesized in five steps. By application of the same or slightly modified procedures additional compounds were prepared. The structures of these compounds are listed in table 1. Radioiodinations of the stannylated precursor were performed with peracetic acid. For chelation with Fu-l77 and Ga-68 the pH of the reaction mixture was adjusted with sodium acetate and heated to 95 °C for 10 min. Stability in human serum was analyzed by precipitation and radio-HPLC analysis of the supernatant.

Reagents

All solvents and non-radioactive reagents were obtained in reagent grade from ABCR (Karlsruhe, Germany), Sigma-Aldrich (Munchen, Germany), Acros Organics (Geel, Belgium) or VWR (Bruchsal, Germany) and were used without further purification. Atto 488 NHS-ester was obtained from AttoTec (Siegen, Germany). 2,2’,2”-(l0-(2-(4-nitrophenyl)oxy)-2- oxoethyl)-l,4,7,l0-tetraazacyclo-dodecane-l,4,7triyl)triacet ic acid (DOTA-PNP) was synthesized following the protocol of Mier et al. (Mier et al., Bioconjug Chem, 2005). The intermediates 6-methoxyquinoline-4-carboxylic acid (7), 5-bromoquinoline-4-carboxylic acid (3) and (.V)- 1 -(2-aminoacctyl)pyrrolidinc-2-carbonitrilc 4-methylbenzenesulfonate were synthesized following the protocols of Jansen et al. (Jansen et al., ACS Med Chem Lett, 2013). The substance (.V)-A-(2-(2-cyanopyrrolidin- l -yl)-2-oxocthyl)-5-bromoquinolinc carboxamide was synthesized by a modified HBTU amidation protocol.

Compound synthesis

Scheme 1 depicts the initial synthesis of FAPI-01 which was achieved by performing a Br/Li- exchange with n-butyllithium at 5-bromoquinolie-4-carboxylic acid (3) and quenching with elemental iodine to obtain iodoquinoline 4. This compound was coupled to the Gly-Pro-CN fragment by HBTU/HOBt-activation to provide non-radioactive reference material of F API-01 (1)·

3 4

Scheme 1. Synthesis of non-radioactive FAPI-01. i) nBuLi, then l ₂ , TH F; ii) H BTU/HOBt, DIPEA, FI-Gly-Pro-CN,

DMF.

For the synthesis of radioactive FAPI-01 (1*), the stannylated precursor 6 was obtained by palladium-catalyzed stannylation of inhibitor 5 in dioxane at 80°C (Scheme 2).

Scheme 2. Synthesis of radioactive FAPI-1 via the stannylated precursor 4. i) (Me ₃ Sn) ₂ ; (PPh ₃ ) ₂ PdCl ; dioxane 80 °C; ii) 1-125 or 1-131; AcOOH; 1 M HCI; MeOH.

To enable radiolabeling by incorporation of radiometals, the chelator DOTA was chemically linked to the basic scaffold of the FAP-inhibitor. As shown by Jansen et al. (Jansen et ah, ACS Med Chem Lett, 2013), modifications at the 6-position of the quinoline-4-carboxylic acid are well tolerated without impairing target affinity and specificity. Therefore, a bifunctional linker was attached to the hydroxyl group of 8 via an ether linkage, leading way to the synthesis shown in Scheme 3. Ready available l-bromo-3-chloropropane was chosen to create a spacer, which is unharmed during the saponification of the simultaneously formed ester bond at the end of the one-pot-process. Compound 9 was converted to the /V-Boc protected quinolinecarboxylic acid 10 which was further coupled to H-Gly-Pro-CN by HBTU. Due to the high hygroscopicity of the free amine, compound 11 was directly converted to FAPI-02 (2) after the Boc-removal, solvent exchange and neutralization of excess -tolucncsulfonic acid.

(5 ^, )-A/-(2-(2-cyanopyrrolidin- 1 -yl)-2-oxoethyl)-5-trimethylstannylquinoline caboxamide (6) 3.88 mg (10.0 qmol) (.V)-A ^/ -(2-(2-cyanopyrrolidin- l -yl)-2-oxocthyl)-5-bromoquinolinc caboxamide, 20 itL (32 mg; 96 ltmol) hexamethylditin and 0.75 mg (1.07 ltmol) bis(triphenylphosphine)palladium(II) dichloride in 1 mL dry dioxane are stirred at 80 °C over night under an inert atmosphere. Volatiles are removed and the residue is taken up in 2 mL 50% acetonitrile/water and filtered through a Cl 8-light cartridge before HPLC-purification. 2.78 mg (5.90 Ltmol; 59%) of the product are obtained after freeze drying.

LC-MS Rt 14.77 min, m/z 473.0786 [M( ¹²⁰ Sn)+H] ⁺

5-iodoquinolie-4-carboxylic acid (4)

5.42 mg (136 pmol) of sodium hydride suspension (60% in mineral oil) are added to an solution of 30.27 mg (120 pmol) 5-bromoquinolie-4-carboxylic acid (3) in 3 mL dry THF under Ar at 0°C. The ice bath is removed and the reaction mixture is cooled to -78 °C before 100 pL (160 pmol) nBuLi (1.6 m in hexanes) are added dropwise. After 15 min 64.71 mg (254 pmol) iodine in 2 mL THF are added dropwise and the reaction is stirred for 30 min at -78 °C before allowed to reach room temperature. After 1 h the reaction is quenched by addition of 1 mL 0.5 M NaHCCL and ca. 30 mg (170 pmol) sodium dithionite to remove excessive iodine. After the removal of THF under reduced pressure the mixture is acidified to pH 2 and extracted three times with ethyl acetate (25 mL). The combined organic phases are evaporated to dryness and purified by HPLC. 18.14 mg (60.7 pmol; 45%) of the title compound are obtained after freeze drying.

¾ NMR (500 MHz, DMSO-d6) 13.95 (br, 0.3H), 8.93 (s, 1H), 8.34 (d, J =7.2 Hz, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.60 (s, 1H), 7.52 (t, J = 7.9 Hz, 1H); ¹³ C NMR (125 MHz, DMSO-d6) 168.8, 150.3, 148.8, 141.3, 130.6, 121.0, 109.5; LC-MS Rt 8.65 min, m/z 299.9383 [M+H] ⁺

(.V)-A-(2-(2-cyanopyrrolidin- 1 -yl)-2-oxoethyl)-5 -trimethylstannylquinoline caboxamide (1 ; FAPI-01)

9.07 mg (23.9 pmol) HBTU in 50 pL DMF are added to a solution of 6.21 mg (20.8 pmol) 5- iodoquinoline-4-carboxylic acid, 7.45 mg (55.2 pmol) HOBt and 10 pL DIPEA in 50 pL DMF. After 15 min (29.9 pmol) (.V)- l -(2-aminoacctyl)pyrrolidinc-2-carbonitrilc 4- methylbenzenesulfonate in 50 pL DMF are added. The reaction is quenched with 850 pL water and purified by HPLC. Freeze drying provides 6.86 mg (15.8 pmol; 76%) of the product.

¾ NMR (600 MHz, DMSO-d6) 9.06, 8.97, 8.33, 8.13, 7.56, 7.51, 4.81, 4.34, 4.06, 3.74, 3.56, 2.21, 2.17, 2.09, 2.05; ¹³ C NMR (150 MHz, DMSO-d6) 167.1, 150.2, 148.8, 145.3, 141.5, 130.7, 125.3, 121.9, 119.3, 92.0, 46.3, 45.4, 42.1, 29.5, 24.9; LC-MS Rt 11.95 min, m/z 435.0102 [M+H] ⁺

6-Hydroxyquinoline-4-carboxylic acid (8)

105 mg (477 pmol) of raw 6-methoxyquinoline-4-carboxylic acid (7) are dissolved in 3 mL of 48% hydrobromic acid in water. The solution is heated to 130 °C for 4 h. The solution is brought to a slightly basic pH with 6 M NaOH after reaching room temperature. 79.2 mg (419 pmol; 88%) of the product are obtained after by HPLC-purification and lyophilization.

¾ NMR (500 MHz, DMSO-d6) 13.65 (br, 0.6H) 10.24 (s, 1H), 8.78 (d, J = 4.4 Hz, 1H), 8.06 (d, J = 2.6 Hz, 1H), 7.95 (d, J = 9.1 Hz, 1H), 7.84 (d, J = 4.4 Hz, 1H), 7.37 (dd, J = 9.1, 2.6 Hz, 1H), ¹³ C NMR (125 MHz, DMSO-d6) 167.7, 156.9, 146.5, 144.1, 133.4, 131.2, 126.2, 122.3, 122.6, 106.5; LC-MS Rt 6.66 min, m/z 190.0415 [M+H] ⁺

6-(3-chloro-l-propoxy)quinoline-4-carboxylic acid (9)

42.4 pL (67.4 mg; 430 pmol) l-bromo-l-chloropropane are added to a suspension of 23.2 mg (123 pmol) 6-hydroxyquinoline-4-carboxylic acid (8) and 190 mg (1.38 pmol) potassium carbonate in 250 pL DMF and heated to 60 °C over night. The reaction mixture is cooled to room temperature, diluted with 500 pL water and 500 pL acetonitrile before 100 pL 6 M NaOH are added. The reaction mixture is directly purified via HPLC (5-40%) after the complete ester hydrolysis is accomplished. 26.45 mg (99.4pmol; 81%) of the product are obtained after lyophilization.

¾ NMR (500 MHz, DMSO-d6) 13.75 (br, 0.4H), 8.88 (d, J = 4.4 Hz, 1H), 8.19 (d, J = 2.0 Hz, 1H), 8.04 (d, J = 9.2 Hz, 1H), 7.94 (d, J = 4.4 Hz, 1H), 7.52 (dd, J = 9.2, 2.0 Hz, 1H), 4.24 (t, J = 5.95 Hz, 2H), 3.85 (t, J = 6.5 Hz, 2H), 2.27 (m, 2H); ¹³ C NMR (125 MHz, DMSO-d6) 167.6, 157.5, 147.6, 144.8, 134.0, 131.2, 125.9, 122.7, 122.2, 104.5, 64.7, 41.9, 31.6; LC-MS Rt 11.46 min, m/z 266.0461 [M+H] ⁺

6-(3-(4-/er/-butoxycarbonylpiperazin-l-yl)-l-propoxy)quinoli ne-4-carboxylic acid (10)

15.13 mg (56.9 pmol) of 6-(3-chloro-l-propoxy)quinoline-4-carboxylic acid (9), 55.43 mg (298 pmol) /V-/er/-butoxycarbonylpiperazine and 51.05 mg (30.8 pmol) potassium iodide are dissolved in 250 pL DMF. The reaction is shaken at 60 °C over night. The resulting suspension is diluted with 750 pL water before the product is purified by HPLC. After freeze drying 28.73 mg (54.3 pmol; 95%) of the product are obtained as the corresponding TFA-salt.

¾ NMR (500 MHz, D ₂ 0) 8.93 (d, J = 5.5 Hz, 1H), 8.17 (d, J = 9.3 Hz, 1H), 7.94 (d, J = 5.5 Hz, 1H), 7.79 (dd, J = 9.3, 2,5 Hz, 1H), 7.65 (d, J = 2.5 Hz, 1H), 4.36 (t, J = 5.6 Hz, 2H), 4.27 (d, J = 13.55 Hz, 2H), 3.67 (d, J = 11.95 Hz), 3.47 (t, J = 15.5 Hz, 2 H), 3.27 (t, J = 12.7 Hz), 3.12 (td, J = 12.2, 2.65 Hz), 2.37 (m2 H), 1.47 (s, 9H); ¹³ C NMR (125 MHz, D2O) 155.5, 153.5, 149.0, 141.4, 134.4, 127.9, 126.6, 122.3, 118.4, 110.0, 105.1, 82.8, 65.5, 54.3, 51.5, 48.6, 40.7, 29.6, 27.4; LC-MS Rt 10.62 min, m/z 416.1997 [M+H] ⁺

(5 ^, )-A-(2-(2-cyanopyrrolidin-l -yl)-2-oxoethyl)-6-(3-(4-/er/-butoxycarbonylpiperazin- 1 -yl)- 1 - propoxy)quinoline-4-carboxamide (11)

9.43 mg (24.9 pmol) HBTU in 50 pL DMF are added to a solution of 10.56 mg (19.9 pmol) 6- (3-(4-/er/-butoxycarbonylpiperazin-l-yl)-l-propoxy)quinoline -4-carboxylic acid (10), 5.38 mg (39.8 pmol) HOBt and 10 pL DIPEA in 50 pL DMF. After 15 min (29.9 pmol) f.S')- 1 -62- ami noacctyl )pyrrol idi nc-2-carbon i tri lc 4-methylbenzenesulfonate in 50 pL DMF are added. The reaction is quenched with 850 pL water and purified by HPLC. Freeze drying provides 12.88 mg (19.4 pmol; 97%) of the title compound.

¾ NMR (500 MHz, DMSO-d6) 9.04 (d, J = 5.5 Hz, 1H), 8.24 (d, J = 9.6 Hz, 1H), 8.10 (d, J = 5.5 Hz, 1H), 7.89 (d, J = 2.3 Hz, 1H), 7.85 (dd, J = 9.6, 2.3 Hz, 1H), 4.84 (t, J = 6 Hz, 1 H), 4.46-4.36 (m, 4H), 4.26 (d, J = 12.0 Hz, 2H), 3.83 (m, 1H), 3.67 (m, 3H), 3.47 (t, J = 7.7 Hz, 2H), 3.27 (br, 2H), 3.11 (t, J = 11.5 Hz), 2.37 (m, 4H), 2.22 (m, 2H), 1.46 (s, 9H); ¹³ C NMR (125 MHz, DMSO-d6) 168.6, 168.0, 159.4, 155.5, 147.7, 141.8, 135.1, 128.2, 127.5, 123.1, 120.0, 119.1, 104.7, 82.9, 66.0, 54.3, 51.5, 47.0, 46.3, 42.3, 29.4, 27.4, 24.7, 23.1; LC-MS Rt 11.81 min, m/z 551.2736 [M+H] ⁺

F API-02

4.85 mg (8.80 mmol) (5 ^, )-/V-(2-(2-cyanopyrrolidin-l-yl)-2-oxoethyl)-6-(3-(4-/ er/- butoxycarbonyl-piperazin-l-yl)-l-propoxy)quino line -4-carboxamide are dissolved in 1 mL acetonitrile and 4.2 mg (22.0 pmol) 4-methylbenzenesulfonic acid monohydrate are added. The reaction is shaken at 45 °C over night, bevore volatiles are removed under reduced pressure. The residue is taken up in 190 pL dimethylformamide and 10 pL (7.3 mg; 72 pmol) triethylamine before 6.77 mg (12.9 mmol) of DOTA- -nitrophcnol ester are added. The reaction mixture is diluted with 1 mL water and purified by HPLC after shaking for two hours. 5.04 mg (6.02 pmol; 68%) are obtained after freeze drying.

LC-MS Rt 8.37 min, m/z 837.3872 [M+H] ⁺

FAPI-02dox

4.10 mg (7.45 pmol) (S)-N-(2-(2-cyanopyrrolidin-l-yl)-2-oxoethyl)-6-(3-(4-tert- butoxycarbonyl-piperazin-l-yl)-l-propoxy)quino line -4-carboxamide are dissolved in 300 pL acetonitrile and 8.87 mg (46.4 pmol) 4-methylbenzenesulfonic acid monohydrate are added. The reaction is shaken at 45 °C for 2 h, before volatiles are removed under reduced pressure. Meanwhile 3.09 mg (8.16 pmol) HBTU in 100 pL DMF are added to 5.98 mg (6.80 pmol) Fmoc-Dox hemiglutarate, 2.07 mg (15.3 pmol) HOBt and 5.00 pL (3.70 mg; 28.7 pmol) DIPEA in 100 pL DMF. After 20 min the solution is added to the dry residue of 4- methylbenzenesulfonic acid and (S)-N-(2-(2-cyanopyrrolidin-l-yl)-2-oxoethyl)-6-(3- piperazin-l-yl-l-propoxy)quinoline-4-carboxamide followed by addition of 100 pL DMF and 20 pL (14.8 mg; 115 pmol) DIPEA. After 120 min 60 pL of piperidine are added and the reaction is purified by RP-HPLC after 15 minutes. 5.63 mg (5.17 pmol; 76%) of the title compound are obtained after freeze-drying.

LC-MS Rt 10.88 min, m/z 1090.3991 [M+H] ⁺

F API- 10

1.06 mg (1.92 pmol) (S)-N-(2-(2-cyanopyrrolidin-l-yl)-2-oxoethyl)-6-(3-(4-tert- butoxycarbonyl-piperazin-l-yl)-l-propoxy)quino line -4-carboxamide are dissolved in 200 pL acetonitrile and 4.31 mg (22.6 pmol) 4-methylbenzenesulfonic acid monohydrate are added. The reaction is shaken at 45 °C for 2 h, before volatiles are removed under reduced pressure. Meanwhile 0.80 mg (2.11 pmol) HBTU in 50 pL DMF are added to 0.41 mg (1.92 pmol) 6-N- maleimidohexanoic acid, and 1.00 pL (0.74 mg; 5.74 pmol) DIPEA in 50 pL DMF. The activated hexanoic acid is added to one half of the residue which is taken up in 50 pL DMF and 5 pL (3.70 mg; 28.7 pmol) DIPEA previously. After 30 min 2.25 mg (1.39 pmol) of the peptide H-Pro-Ala-Ala-Lys-Arg-Val-Lys-Leu-Asp-Lys(DOTA)-Cys-OH is added. After completion the mixture is purified by HPLC. 1.43 mg (0.63 pmol; 66%) are obtained after freeze-drying. LC-MS Rt 9.58 min, m/z 753.0461 [M+3H] ³⁺

Compound analysis

Reverse-phase high-performance liquid chromatography (RP-HPLC) was conducted using linear gradients of acetonitrile in water (0-100% acetonitrile in 5 min; 0.1% TFA; flowrate 2 mL/min) on a Chromolith Performance RP-l8e column (100 x 3 mm; Merck KGaA Darmstadt, Germany). UV-absorbance was detected at 214 nm. An additional g-detector was used for the HPLC-analysis of radioactive compounds. HPLC-MS characterization was performed on an ESI mass spectrometer (Exactive, Thermo Fisher Scientific, Waltham, MA, USA) connected to an Agilent 1200 HPLC system with a Hypersil Gold C18 1.9 pm column (200 x 2.1 mm; 0- 100% acetonitrile in 20 min; flowrate 200 pL/min). Analytical Radio-HPLC was performed using a Chromolith Performance RP-l8e column (l00><3mm; Merck; 0-30% acetonitrile in 10 min; flowrate 2 mL/min). HPLC-purifications were performed on a LaPrep Pl lO-System (Knauer, Berlin, Germany) and a Reprosil Pur 120 column (Cl8-aq 5 pm 250 x 25mm; Dr. Maisch, Ammerbuch-Entringen, Germany). The water/acetonitrile-gradient (15 or 25 min; 0.1% TFA; flowrate 20 mL/min) was modified for the individual products.

Example 2: In vitro characterization of FAPI derivatives

In vitro binding studies were performed using the human tumor cell lines BxPC3, Capan-2, MCF-7 (purchased from Sigma Aldrich Chemie GmbH) and SK-LMS-l (purchased from ATCC) as well as stably transfected FAP-cell lines HT-1080-FAP, HEK-muFAP and the CD26 expressing cell line HEK-CD26 (obtained from Stefan Bauer, NCT Heidelberg). All cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum at 37°C/5% carbon dioxide. For fluorescence internalization experiments, cells were seeded on coverslips and stained with FAPI-02-Atto488 and DAPI for cell nucleus staining. Images were acquired on a laser scanning confocal microscope using a 63x oil immersion objective. Radioligand binding studies were performed using HT-1080-FAP cells. The radiolabeled compound was added to the cell culture and incubated for different time intervals ranging from 10 min to 24 h. Competition experiments were performed by simultaneous exposure to unlabeled (10 ⁵ M to lO ⁹ M) and radiolabeled compound for 60 min. For efflux experiments, radioactive medium was removed after incubation for 60 min and replaced by non-radioactive medium for time intervals ranging from 1 to 24 h. For internalization experiments, surface bound activity was removed by incubating the cells with 1 M glycine -HC1 buffer for 10 min. The radioactivity was measured using a g-counter, normalized to 1 mio cells and calculated as percentage of applied dose (%ID).

Cell staining and microscopy

For internalization experiments HT- 1080-FAP and HEK muFAP cells were seeded on uncoated coverslips in a 24-well plate and cultivated in culture medium containing 10% fetal calf serum to a final confluence of approx. 80-90%. The medium was removed and cells were washed with 0.5 mL PBS pH 7.4 for 2 times. FAPI-02-Atto488 (20 mM in DMEM) was added to the cells and incubated for 2 hrs at 37°C. Cells were washed with 0.5 mL PBS pH 7.4 for 3 times and fixed with paraformaldehyde (2% in PBS) for 15 min. The overgrown coverslips were placed on microscope slides using mounting medium containing DAPI for cell nucleus staining (Fluoroshield, Sigma- Aldrich). Images were acquired on a laser scanning confocal microscope (Zeiss LSM 700; Zeiss, Oberkochen, Germany) using the Zeiss Plan-Apochromat 63x/l .4 Oil DIC III immersion objective at xy pixel settings of 0.099 x 0.099 pm and 1 Airy unit pinhole size for each fluorophore used (488 nm for FAPI-02-Atto488, 405 nm for DAPI). The pictures were processed consistently using the ZEN 2008 software and ImageJ.

F API-02 shows enhanced binding and uptake to human FAP-a as compared to FAPI-01.

To avoid rapid loss of activity of FAPI-01 due to enzymatic deiodination, the non-halogen derivative FAPI-02 was designed in which the FAP -binding moiety is chemically linked to the chelator DOTA. In addition to the resulting enhanced stability, this modification offers the possibility to easily incorporate either diagnostic or therapeutic radionuclides, allowing FAPI- 02 to be used as a theranostic compound. Similar to its iodized analogue, FAPI-02 specifically binds to human and murine FAP-a (ICso human FAP-a = 21 nM) expressing cells without addressing CD26 (%ID = 0.13 ± 0.01 %; Figure 1A). FAPI-02 internalizes rapidly into FAP-a expressing cells (20.15 ± 1.74 %ID after 60 min, of which 96 % internalized; Figure 1B), showing more stable and higher uptake rates in the course of time. Compared to the binding of F API-01 after 10 min of incubation, only 5 % of the activity remains after 24 h. In contrast, 34 % of the initial radioactivity of FAPI-02 is detected after 24 h of incubation. Efflux experiments demonstrate that FAPI-02 gets eliminated significantly slower than FAPI-01 , showing retention of 12 % of the originally accumulated radioactivity after 24 h (FAPI-01 1.1 % ID after 24 h; Figure 1E).

Robust internalization of FAPI-02 into human and murine FAP-a expressing cells was confirmed by fluorescence laser scanning microscopy. To this end, HT-1080-FAP and HEK- muFAP cells were stained with a fluorescently labeled FAPI-02 derivative (FAPI-02-Atto488) for 1 to 2 hrs. As shown in Figure 1D, the compound gets completely internalized and accumulates in the inner of FAP-a expressing cells whereas no uptake is detectable in FAP-a negative HEK-CD26 cells. Design of FAPI derivatives with enhanced binding properties and pharmacokinetics

Further variants of FAPI-02 were designed to increase tumor retention time, aiming for the development of a FAP-targeting agent. The variants FAPI-03 to FAPI- 15 have been characterized with respect to target binding, internalization rate and target specificity. The results are shown in Figure 2.

MTT assay for colorimetric assessment of cytotoxic effects of Doxorubicin conjugated to FAPI-02 (FAPI-02-Dox) vs. unconjugated Doxorubicin

To determine the cytotoxic potential of FAPI-02-Dox, a colorimetric assay using MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is performed. MTT is a tetrazolium dye which gets reduced by cellular enzymes resulting in the formation of the insoluble purple dye formazan. The amount of the formed dye can be quantified by UV spectroscopy and reflects the number of viable cells present. To perform the assay, HT-1080-FAP cells are seeded in culture medium (DMEM containing 10% fetal calf serum) in 96-well plates and grown for two days to a final confluence of 80-90%. The culture medium is removed and the cells incubated with FAPI-02-Dox and unconjugated Doxorubicin, which serves as a positive control, for 1 to 3 days at 37°C. Both compounds are added in culture medium in six different concentrations (0.1, 1, 10, 100, 1000, 10000 nM resp.). After incubation the medium is removed, MTT added in a final concentration of 0.5 mg/ml and incubated for 3 h at 37°C. After removing the dye solution, DMSO is added and incubated for 30 min in the dark to dissolve the dye crystals. Cell viability is determined by measuring absorption at 560 nm using a plate reader (Tecan Group Ltd., Mannedorf, Switzerland).

Further assays for characterization of FAPI derivatives

Tissue explants (as described previously in Halama et al., Cancer Cell, 2016) are being used for characterization of the invention. Histology for whole slide quantification of immune cells and measurements of cytokines within tissues are used to evaluate the invention. Enhanced infiltration rates of immune cells and modulation of the microenvironment are observed for all embodiments of the invention.